Engineered CRISPR-Cas9 nucleases

ABSTRACT

Engineered CRISPR-Cas9 nucleases with improved specificity and their use in genomic engineering, epigenomic engineering, genome targeting, and genome editing.

CLAIM OF PRIORITY

This application claims priority under 35 USC §119(e) to U.S. PatentApplication Ser. No. 62/211,553, filed on Aug. 28, 2015; 62/216,033,filed on Sep. 9, 2015; and 62/258,280, filed on Nov. 20, 2015. Theentire contents of the foregoing are hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant Nos. DP1GM105378 and RO1 GM088040 awarded by the National Institutes of Health.The Government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 4, 2016, isnamed 29539-0189001_SL.txt and is 129,880 bytes in size.

TECHNICAL FIELD

The invention relates, at least in part, to engineered ClusteredRegularly Interspaced Short Palindromic Repeats(CRISPRs)/CRISPR-associated protein 9 (Cas9) nucleases with altered andimproved target specificity and their use in genomic engineering,epigenomic engineering, genome targeting, genome editing, and in vitrodiagnostics.

BACKGROUND

CRISPR-Cas9 nucleases enable efficient genome editing in a wide varietyof organisms and cell types (Sander & Joung, Nat Biotechnol 32, 347-355(2014); Hsu et al., Cell 157, 1262-1278 (2014); Doudna & Charpentier,Science 346, 1258096 (2014); Barrangou & May, Expert Opin Biol Ther 15,311-314 (2015)). Target site recognition by Cas9 is programmed by achimeric single guide RNA (sgRNA) that encodes a sequence complementaryto a target protospacer (Jinek et al., Science 337, 816-821 (2012)), butalso requires recognition of a short neighboring PAM (Mojica et al.,Microbiology 155, 733-740 (2009); Shah et al., RNA Biol 10, 891-899(2013); Jiang et al., Nat Biotechnol 31, 233-239 (2013); Jinek et al.,Science 337, 816-821 (2012); Sternberg et al., Nature 507, 62-67(2014)).

SUMMARY

As described herein, Cas9 Proteins can be engineered to show increasedspecificity, theoretically by reducing the binding affinity of Cas9 forDNA. Thus, described herein are a number of Cas9 variants that haveincreased specificity (i.e., induce substantially fewer off targeteffects) as compared to the wild type protein, as well as methods ofusing them.

In a first aspect, the invention provides isolated Streptococcuspyogenes Cas9 (SpCas9) proteins with mutations at one, two, three, four,five, six or all seven of the following positions: L169A, Y450, N497,R661, Q695, Q926, and/or D1135E e.g., comprising a sequence that is atleast 80% identical to the amino acid sequence of SEQ ID NO:1 withmutations at one, two, three, four, five, six, or seven of the followingpositions: L169, Y450, N497, R661, Q695, Q926, D1135E, and optionallyone or more of a nuclear localization sequence, cell penetrating peptidesequence, and/or affinity tag. A mutation alters the amino acid to anamino acid other than the native amino acid (e.g., 497 is anything butN). In preferred embodiments the mutation changes the amino acid to anyamino acid other than the native one, arginine or lysine; in someembodiments, the amino acid is alanine.

In some embodiments, the variant SpCas9 proteins comprise one, two,three, or all four of the following mutations: N497A, R661A, Q695A, andQ926A.

In some embodiments, the variant SpCas9 proteins comprise mutations atQ695 and/or Q926, and optionally one, two, three, four or all five ofL169, Y450, N497, R661 and D1135Ee.g., including but not limited toY450A/Q695A, L169A/Q695A, Q695A/Q926A, Q695A/D1135E, Q926A/D1135E,Y450A/D1135E, L169A/Y450A/Q695A, L169A/Q695A/Q926A, Y450A/Q695A/Q926A,R661A/Q695A/Q926A, N497A/Q695A/Q926A, Y450A/Q695A/D1135E,Y450A/Q926A/D1135E, Q695A/Q926A/D1135E, L169A/Y450A/Q695A/Q926A,L169A/R661A/Q695A/Q926A,Y450A/R661A/Q695A/Q926A,N497A/Q695A/Q926A/D1135E, R661A/Q695A/Q926A/D1135E, andY450A/Q695A/Q926A/D1135E.

In some embodiments, the variant SpCas9 proteins comprise mutations atR63; R78; H160; K163; R165; L169; R403; N407; Y450; M495; N497; K510;Y515; W659; R661; M694; Q695; H698; A728; Q926; K1107; E1108; S1109;K1113; R1114; S1116; K1118; D1135; S1136; K1153; K1155; K1158; K1200;Q1221; K1289; R1298; K1300; K1325; K1334; T1337 and/or S1216.

In some embodiments, the variant SpCas9 proteins also comprise one ormore of the following mutations: R63A; R78A; R165A; R403A; N407A; N497A;Y450A; K510A; Y515A; R661A; Q695A; Q926A; K1107A; E1108A; S1109A;K1113A; R1114A; S1116A; K1118A; D1135A; S1136A; K1153A; K1155A; K1158A;K1200A; Q1221A; K1289A; R1298A; K1300A; K1325A; K1334A; T1337A and/orS1216A. In some embodiments, variant SpCas9 proteins comprise one ormore of the following additional mutations: R63A, R66A, R69A, R70A,R71A, Y72A, R74A, R75A, K76A, N77A, R78A, R115A, H160A, K163A, R165A,L169A, R403A,T404A, F405A, N407A, R447A, N497A, 1448A, Y450A, S460A,M495A, K510A, Y515A, R661A, M694A, Q695A, H698A, Y1013A, V1015A, R1122A,K1123A, K1124A, K1158A, K1185A, K1200A, S1216A, Q1221A, K1289A, R1298A,K1300A, K1325A, R1333A, K1334A, R1335A, and T1337A.

In some embodiments, the variant SpCas9 proteins comprise multiplesubstitution mutations: N497/R661/Q695/Q926 (quadruple variant mutants);Q695/Q926 (double mutant); R661/Q695/Q926 and N497/Q695/Q926 (triplemutants). In some embodiments, the additional substitution mutations atL169, Y450 and/or D1135 might be added to these double-, triple, andquadruple mutants or added to single mutants bearing substitutions atQ695 or Q926. In some embodiments, the mutants have alanine in place ofthe wild type amino acid. In some embodiments, the mutants have anyamino acid other than arginine or lysine (or the native amino acid).

In some embodiments, the variant SpCas9 proteins also comprise one ormore mutations that decrease nuclease activity selected from the groupconsisting of mutations at D10, E762, D839, H983, or D986; and at H840or N863.

In some embodiments, the mutations are: (i) D10A or D10N, and (ii)H840A, H840N, or H840Y.

In some embodiments, the SpCas9 variants can also include one of thefollowing sets of mutations: D1135V/R1335Q/T1337R (VQR variant);D1135E/R1335Q/T1337R (EQR variant); D1135V/G1218R/R1335Q/T1337R (VRQRvariant); or D1135V/G1218R/R1335E/T1337R (VRER variant).

Also provided herein are isolated Staphylococcus aureus Cas9 (SaCas9)protein, with mutations at one, two, three, four, five, or six of thefollowing positions: Y211, W229, R245, T392, N419, R654, e.g.,comprising a sequence that is at least 80% identical to the amino acidsequence of SEQ ID NO:1 with mutations at one, two, three, four, orfive, or six of the following positions: Y211, W229, R245, T392, N419,R654, and optionally one or more of a nuclear localization sequence,cell penetrating peptide sequence, and/or affinity tag. In someembodiments, the SaCas9 to variants described herein include the aminoacid sequence of SEQ ID NO:2, with mutations at one, two, three, four,five, or all six of the following positions: Y211, W229, R245, T392,N419, and/or R654. The isolated protein of claim 8, comprising one ormore of the following mutations: Y211A, W229, R245A, T392A, N419A,and/or R654A.

In some embodiments, the variant SaCas9 proteins comprise mutations atN419 and/or R654, and optionally one, two, three or four of theadditional mutations Y211, W229, R245 and T392, e.g., including but notlimited to N419A/R654A, Y211A/R654A, W229A/R654A, Y211A/R245A/R654A,Y211A/R245A/N419A, Y211A/N419A/R654A, R245A/N419A/R654A,T392A/N419A/R654A, R245A/T392A/N419A/R654A, Y211A/R245A/N419A/R654A,W229A/R245A/N419A/R654A, Y211A/R245A/T392A/N419A/R654A, andY211A/W229A/R245A/N419A/R654A.

In some embodiments, the variant SaCas9 proteins comprise mutations atY211; W229; Y230; R245; T392; N419; L446; Y651; R654; D786; T787; Y789;T882; K886; N888; 889; L909; N985; N986; R991; R1015; N44; R45; R51;R55; R59; R60; R116; R165; N169; R208; R209; Y211; T238; Y239; K248;Y256; R314; N394; Q414; K57; R61; H111; K114; V164; R165; L788; 5790;R792; N804; Y868; K870; K878; K879; K881; Y897; R901; and/or K906.

In some embodiments, the variant SaCas9 proteins comprise one or more ofthe following mutations: Y211A; W229A; Y230A; R245A; T392A; N419A;L446A; Y651A; R654A; D786A; T787A; Y789A; T882A; K886A; N888A; A889A;L909A; N985A; N986A; R991A; R1015A; N44A; R45A; R51A; R55A; R59A; R60A;R116A; R165A; N169A; R208A; R209A; T238A; Y239A; K248A; Y256A; R314A;N394A; Q414A; K57A; R61A; H111A; K114A; V164A; R165A; L788A; S790A;R792A; N804A; Y868A; K870A; K878A; K879A; K881A; Y897A; R901A; K906A.

In some embodiments, variant SaCas9 proteins comprise one or more of thefollowing additional mutations: Y211A, W229A, Y230A, R245A, T392A,N419A, L446A, Y651A, R654A, D786A, T787A, Y789A, T882A, K886A, N888A,A889A, L909A, N985A, N986A, R991A, R1015A, N44A, R45A, R51A, R55A, R59A,R60A, R116A, R165A, N169A, R208A, R209A, T238A, Y239A, K248A, Y256A,R314A, N394A, Q414A, K57A, R61A, H111A, K114A, V164A, R165A, L788A,S790A, R792A, N804A, Y868A, K870A, K878A, K879A, K881A, Y897A, R901A,K906A.

In some embodiments, the variant SaCas9 proteins comprise multiplesubstitution mutations: R245/T392/N419/R654 and Y221/R245/N419/R654(quadruple variant mutants); N419/R654, R245/R654, Y221/R654, andY221/N419 (double mutants); R245/N419/R654, Y211/N419/R654, andT392/N419/R654 (triple mutants). In some embodiments the mutants containalanine in place of the wild type amino acid.

In some embodiments, the variant SaCas9 proteins also comprise one ormore mutations that decrease nuclease activity selected from the groupconsisting of mutations at D10, E477, D556, H701, or D704; and at H557or N580. In some embodiments, the mutations are: (i) D10A or D10N, (ii)H557A, H557N, or H557Y, (iii) N580A, and/or (iv) D556A.

In some embodiments, the variant SaCas9 proteins comprise one or more ofthe following mutations: E782K, K929R, N968K, or R1015H. Specifically,E782K/N968K/R1015H (KKH variant); E782K/K929R/R1015H (KRH variant); orE782K/K929R/N968K/R1015H (KRKH variant).

In some embodiments, the variant Cas9 proteins include mutations to oneor more of the following regions to increase specificity:

Functional Region SpCas9 SaCas9 Residues contacting L169; Y450; M495;Y211; W229; Y230; the DNA of the N497; W659; R661; R245; T392; N419;spacer region M694; Q695; H698; L446; Y651; R654 A728; Q926; E1108;V1015 Residues contacting R71; Y72; R78; R165; D786; T787; Y789; the DNAof the R403; T404; F405; T882; K886; N888; PAM region K1107; S1109;R1114; A889; L909; N985; (including direct S1116; K1118; D1135; N986;R991; R1015 PAM contacts) S1136; K1200; S1216; E1219; R1333; R1335;T1337 Residues contacting Y72; R75; K76; L101; N44; R45; R51; the RNA ofthe S104; F105; R115; R55; R59; R60; spacer region H116; I135; H160;R116; R165; K163; Y325; H328; N169; R208; R209; R340; F351; D364; Y211;T238; Y239; Q402; R403; I1110; K248; Y256; R314; K1113; R1122; Y1131N394; Q414 Residues contacting R63; R66; R70; R71; K57; R61; H111; theRNA of R74; R78; R403; T404; K114; V164; R165; the repeat/anti- N407;R447; I448; L788; S790; R792; repeat region Y450; K510; N804; Y868;Y515; R661; K870; K878; K879; V1009; Y1013 K881; Y897; R901; K906Residues contacting K30; K33; N46; R40; R47; K50; R54; R58; the RNA K44;E57; T62; R69; H62; R209; E213; stem loops N77; L455; S460; R467; S219;R452; T472; I473; H721; K742; K459; R774; N780; K1097; V1100; T1102;R781; L783 F1105; K1123; K1124; E1225; Q1272; H1349; S1351; Y1356

Also provided herein are fusion proteins comprising the isolated variantCas9 proteins described herein fused to a heterologous functionaldomain, with an optional intervening linker, wherein the linker does notinterfere with activity of the fusion protein. In preferred embodiments,the heterologous functional domain acts on DNA or protein, e.g., onchromatin. In some embodiments, the heterologous functional domain is atranscriptional activation domain. In some embodiments, thetranscriptional activation domain is from VP64 or NF-κB p65. In someembodiments, the heterologous functional domain is a transcriptionalsilencer or transcriptional repression domain. In some embodiments, thetranscriptional repression domain is a Kruppel-associated box (KRAB)domain, ERF repressor domain (ERD), or mSin3A interaction domain (SID).In some embodiments, the transcriptional silencer is HeterochromatinProtein 1 (HP1), e.g., HP1α or HP1β. In some embodiments, theheterologous functional domain is an enzyme that modifies themethylation state of DNA. In some embodiments, the enzyme that modifiesthe methylation state of DNA is a DNA methyltransferase (DNMT) or theentirety or the dioxygenase domain of a TET protein, e.g., a catalyticmodule comprising the cysteine-rich extension and the 2OGFeDO domainencoded by 7 highly conserved exons, e.g., the Tet1 catalytic domaincomprising amino acids 1580-2052, Tet2 comprising amino acids 1290-1905and Tet3 comprising amino acids 966-1678. In some embodiments, the TETprotein or TET-derived dioxygenase domain is from TET1. In someembodiments, the heterologous functional domain is an enzyme thatmodifies a histone subunit. In some embodiments, the enzyme thatmodifies a histone subunit is a histone acetyltransferase (HAT), histonedeacetylase (HDAC), histone methyltransferase (HMT), or histonedemethylase. In some embodiments, the heterologous functional domain isa biological tether. In some embodiments, the biological tether is MS2,Csy4 or lambda N protein. In some embodiments, the heterologousfunctional domain is FokI.

Also provided herein are nucleic acids, isolated nucleic acids encodingthe variant Cas9 proteins described herein, as well as vectorscomprising the isolated nucleic acids, optionally operably linked to oneor more regulatory domains for expressing the variant Cas9 proteinsdescribed herein. Also provided herein are host cells, e.g., bacterial,yeast, insect, or mammalian host cells or transgenic animals (e.g.,mice), comprising the nucleic acids described herein, and optionallyexpressing the variant Cas9 proteins described herein.

Also provided herein are methods of altering the genome of a cell, byexpressing in the cell isolated variant Cas9 proteins as describedherein, and at least one guide RNA having a region complementary to aselected portion of the genome of the cell with optimal nucleotidespacing at the genomic target site.

Also provided herein are methods of altering the genome of a cell, byexpressing in the cell an isolated variant Cas9 protein describedherein, and a guide RNA having a region complementary to a selectedportion of the genome of the cell with optimal nucleotide spacing at thegenomic target site.

Also provided herein are isolated nucleic acids encoding theCas9variants, as well as vectors comprising the isolated nucleic acids,optionally operably linked to one or more regulatory domains forexpressing the variants, and host cells, e.g., mammalian host cells,comprising the nucleic acids, and optionally expressing the variantproteins.

Also provided herein are methods for altering, e.g., selectivelyaltering, the genome of a cell by contacting the cell with, orexpressing in the cell, a variant protein as described herein, and aguide RNA having a region complementary to a selected portion of thegenome of the cell. In some embodiments, the isolated protein or fusionprotein comprises one or more of a nuclear localization sequence, cellpenetrating peptide sequence, and/or affinity tag.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-E|Identification and characterization of SpCas9 variantsbearing mutations in residues that form non-specific DNA contacts. a,Schematic depicting wild-type SpCas9 recognition of the target DNA:sgRNAduplex, based on PDB 4OOG and 4UN3 (adapted from refs. 31 and 32,respectively). b, Characterization of SpCas9 variants that containalanine substitutions in positions that form hydrogen bonds to the DNAbackbone. Wild-type SpCas9 and variants were assessed using the humancell EGFP disruption assay when programmed with a perfectly matchedsgRNA or four other sgRNAs that encode mismatches to the target site.Error bars represent s.e.m. for n=3; mean level of background EGFP lossrepresented by red dashed line (for this panel and panel c). c and d,On-target activities of wild-type SpCas9 and SpCas9-HF1 across 24 sitesassessed by EGFP disruption assay (panel c) and 13 endogenous sites byT7E1 assay (panel d). Error bars represent s.e.m. for n=3. e, Ratio ofon-target activity of SpCas9-HF1 to wild-type SpCas9 (from panels c andd). The median and in

FIG. 2A-C|Genome-wide specificities of wild-type SpCas9 and SpCas9-HF1with sgRNAs for standard target sites. a, Off-target sites of wild-typeSpCas9 and SpCas9-HF1 with eight sgRNAs targeted to endogenous humangenes, as determined by GUIDE-seq. Read counts represent a measure ofcleavage frequency at a given site; mismatched positions within thespacer or PAM are highlighted in color. b, Summary of the total numberof genome-wide off-target sites identified by GUIDE-seq for wild-typeSpCas9 and SpCas9-HF1 from the eight sgRNAs used in panel a. c,Off-target sites identified for wild-type SpCas9 and SpCas9-HF1 for theeight sgRNAs, binned according to the total number of mismatches (withinthe protospacer and PAM) relative to the on-target site.

FIG. 3A-C|Validation of SpCas9-HF1 specificity improvements by targeteddeep sequencing of off-target sites identified by GUIDE-seq. a, Meanon-target percent modification determined by deep sequencing forwild-type SpCas9 and SpCas9-HF1 with six sgRNAs from FIG. 2. Error barsrepresent s.e.m. for n=3. b, Percentage of deep sequenced on-targetsites and GUIDE-seq detected off-target sites that contain indelmutations. Triplicate experiments are plotted for wild-type SpCas9,SpCas9-HF1, and control conditions. Filled circles below the x-axisrepresent replicates for which no insertion or deletion mutations wereobserved. Off-target sites that could not be amplified by PCR are shownin red text with an asterisk. Hypothesis testing using a one-sidedFisher exact test with pooled read counts found significant differences(p<0.05 after adjusting for multiple comparisons using theBenjamini-Hochberg method) for comparisons between SpCas9-HF1 and thecontrol condition only at EMX1-1 off-target 1 and FANCF-3 off-target 1.Significant differences were also found between wild-type SpCas9 andSpCas9-HF1 at all off-target sites, and between wild-type SpCas9 and thecontrol condition at all off-target sites except RUNX1-1 off-target 2.c, Scatter plot of the correlation between GUIDE-seq read counts (fromFIG. 2a ) and mean percent modification determined by deep sequencing aton- and off-target cleavage sites with wild-type SpCas9.

FIG. 4A-C|Genome-wide specificities of wild-type SpCas9 and SpCas9-HF1with sgRNAs for non-standard, repetitive sites. a, GUIDE-seq specificityprofiles of wild-type SpCas9 and SpCas9-HF1 using two sgRNAs known tocleave large numbers of off-target sites4,8. GUIDE-seq read countsrepresent a measure of cleavage efficiency at a given site; mismatchedpositions within the spacer or PAM are highlighted in color; red circlesindicate sites likely to have the indicated bulge12 at the sgRNA-DNAinterface; blue circles indicate sites that may have an alternativegapped alignment relative to the one shown (see FIG. 8). b, Summary ofthe total number of genome-wide off-target sites identified by GUIDE-seqfor wild-type SpCas9 and SpCas9-HF1 from the two sgRNAs used in panel a.c, Off-target sites identified with wild-type SpCas9 or SpCas9-HF1 forVEGFA sites 2 and 3, binned according to the total number of mismatches(within the protospacer and PAM) relative to the on-target site.Off-target sites marked with red circles in panel a are not included inthese counts; sites marked with blue circles in panel a are counted withthe number of mismatches in the non-gapped alignment.

FIG. 5A-D|Activities of SpCas9-HF1 derivatives bearing additionalsubstitutions. a, Human cell EGFP disruption activities of wild-typeSpCas9, SpCas9-HF1, and SpCas9-HF1-derivative variants with eightsgRNAs. SpCas9-HF1 harbors N497A, R661A, Q695, and Q926A mutations;HF2=HF1+D1135E; HF3=HF1+L169A; HF4=HF1+Y450A. Error bars represents.e.m. for n=3; mean level of background EGFP loss represented by thered dashed line. b, Summary of the on-target activity when usingSpCas9-HF variants compared to wild-type SpCas9 with the eight sgRNAsfrom panel a. The median and interquartile range are shown; the intervalshowing >70% of wild-type activity is highlighted in green. c, Meanpercent modification by SpCas9 and HF variants at the FANCF site 2 andVEGFA site 3 on-target sites, as well as off-target sites from FIGS. 2aand 4a resistant to the effects of SpCas9-HF1. Percent modificationdetermined by T7E1 assay; background indel percentages were subtractedfor all experiments. Error bars represent s.e.m. for n=3. d, Specificityratios of wild-type SpCas9 and HF variants with the FANCF site 2 orVEGFA site 3 sgRNAs, plotted as the ratio of on-target to off-targetactivity (from panel c).

FIG. 6A-B|SpCas9 interaction with the sgRNA and target DNA. a, Schematicillustrating the SpCas9:sgRNA complex, with base pairing between thesgRNA and target DNA. b, Structural representation of the SpCas9:sgRNAcomplex bound to the target DNA, from PDB: 4UN3 (ref 32). The fourresidues that form hydrogen bond contacts to the target-strand DNAbackbone are highlighted in blue; the HNH domain is hidden forvisualization purposes.

FIG. 7A-D|On-target activity comparisons of wild-type and SpCas9-HF1with various sgRNAs used for GUIDE-seq experiments. a and c, MeanGUIDE-seq tag integration at the intended on-target site for GUIDE-seqexperiments shown in FIGS. 2a and 4a (panels a and c, respectively),quantified by restriction fragment length polymorphism assay. Error barsrepresent s.e.m. for n=3. b and d, Mean percent modification at theintended on-target site for GUIDE-seq experiments shown in FIGS. 2a and4a (panels b and d, respectively), detected by T7E1 assay. Error barsrepresent s.e.m. for n=3.

FIG. 8|Potential alternate alignments for VEGFA site 2 off-target sites.Ten VEGFA site 2 off-target sites identified by GUIDE-seq (left) thatmay potentially be recognized as off-target sites that contain singlenucleotide gaps (ref 12) (right), aligned using Geneious (ref 45)version 8.1.6.

FIG. 9|Activities of wild-type SpCas9 and SpCas9-HF1 with truncatedsgRNAs14. EGFP disruption activities of wild-type SpCas9 and SpCas9-HF1using full-length or truncated sgRNAs targeted to four sites in EGFP.Error bars represent s.e.m. for n=3; mean level of background EGFP lossin control experiments is represented by the red dashed line

FIG. 10|Wild-type SpCas9 and SpCas9-HF1 activities with sgRNAs bearing5′-mismatched guanine bases. EGFP disruption activities of wild-typeSpCas9 and SpCas9-HF1 with sgRNAs targeted to four different sites. Foreach target site, sgRNAs either contain the matched non-guanine 5′-baseor a 5′-guanine that is intentionally mismatched.

FIG. 11|Titrating the amount of wild-type SpCas9 and SpCas9-HF1expression plasmids. Human cell EGFP disruption activities fromtransfections with varying amounts of wild-type and SpCas9-HF1expression plasmids. For all transfections, the amount ofsgRNA-containing plasmid was fixed at 250 ng. Two sgRNAs targetingseparate sites were used; Error bars represent s.e.m. for n=3; meanlevel of background EGFP loss in negative controls is represented by thered dashed line.

FIG. 12A-D|Altering the PAM recognition specificity of SpCas9-HF1. a,Comparison of the mean percent modification of on-target endogenoushuman sites by SpCas9-VQR (ref 15) and an improved SpCas9-VRQR using 8sgRNAs, quantified by T7E1 assay. Both variants are engineered torecognize an NGAN PAM. Error bars represent s.e.m. for n=2 or 3. b,On-target EGFP disruption activities of SpCas9-VQR and SpCas9-VRQRcompared to their -HF1 counterparts using eight sgRNAs. Error barsrepresent s.e.m. for n=3; mean level of background EGFP loss in negativecontrols represented by the red dashed line. c, Comparison of the meanon-target percent modification by SpCas9-VQR and SpCas9-VRQR compared totheir -HF1 variants at eight endogenous human gene sites, quantified byT7E1 assay. Error bars represent s.e.m. for n=3; ND, not detectable. d,Summary of the fold-change in on-target activity when using SpCas9-VQRor SpCas9-VRQR compared to their corresponding -HF1 variants (frompanels b and c). The median and interquartile range are shown; theinterval showing >70% of wild-type activity is highlighted in green.

FIGS. 13A-B|Genome-wide specificities of SpCas9-HF1, -HF2, and -HF4 withsgRNAs that have off-target sites resistant to the effects ofSpCas9-HF1. a, Mean GUIDE-seq tag integration at the intended on-targetsite for GUIDE-seq experiments in panel b.SpCas9-HF1=N497A/R661A/Q695A/Q926A; HF2=HF1+D1135E; HF4=HF1+Y450A. Errorbars represent s.e.m. for n=3. b, GUIDE-seq identified off-target sitesof SpCas9-HF1, -HF2, or -HF4 with either the FANCF site 2 or VEGFA site3 sgRNAs. Read counts represent a measure of cleavage frequency at agiven site; mismatched positions within the spacer or PAM arehighlighted in color. The fold-improvement in off-target discriminationwas calculated by normalizing the off-target read counts for anSpCas9-HF variant to the read counts at the on-target site prior tocomparison between SpCas9-HF variants.

FIG. 14|Structural comparison of SpCas9 (top) and SaCas9 (bottom)illustrating the similarity between the positions of the mutations inthe quadruple mutant constructs (shown in yellow sphere representation).Also, shown in pink sphere representation are other residues thatcontact the DNA backbone.

DETAILED DESCRIPTION

A limitation of the CRISPR-Cas9 nucleases is their potential to induceundesired off-target mutations (see, for example, Tsai et al., NatBiotechnol. 2015), in some cases with frequencies rivaling thoseobserved at the intended on-target site (Fu et al., Nat Biotechnol.2013). Previous work with CRISPR-Cas9 nucleases has suggested thatreducing the number of sequence-specific interactions between the guideRNA (gRNA) and the spacer region of a target site can reduce mutageniceffects at off-target sites of cleavage in human cells (Fu et al., NatBiotechnol. 2014).

This was earlier accomplished by truncating gRNAs at their 5′ ends by 2or 3 nts and it was hypothesized that the mechanism of this increasedspecificity was a decrease in the interaction energy of the gRNA/Cas9complex so that it was poised with just enough energy to cleave theon-target site, making it less likely to have enough energy to cleaveoff-target sites where there would presumably be an energetic penaltydue to mismatches in the target DNA site (WO2015/099850).

It was hypothesized that off-target effects of SpCas9 might be minimizedby decreasing non-specific interactions with its target DNA site.SpCas9-sgRNA complexes cleave target sites composed of an NGG PAMsequence (recognized by SpCas9) (Deltcheva, E. et al. Nature 471,602-607 (2011); Jinek, M. et al. Science 337, 816-821 (2012); Jiang, W.,et al., Nat Biotechnol 31, 233-239 (2013); Sternberg, S. H., et al.,Nature 507, 62-67 (2014)) and an adjacent 20 by protospacer sequence(which is complementary to the 5′ end of the sgRNA) (Jinek, M. et al.Science 337, 816-821 (2012); Jinek, M. et al. Elife 2, e00471 (2013);Mali, P. et al., Science 339, 823-826 (2013); Cong, L. et al., Science339, 819-823 (2013)). It was previously theorized that the SpCas9-sgRNAcomplex may possess more energy than is needed for recognizing itsintended target DNA site, thereby enabling cleavage of mismatchedoff-target sites (Fu, Y., et al., Nat Biotechnol 32, 279-284 (2014)).One can envision that this property might be advantageous for theintended role of Cas9 in adaptive bacterial immunity, giving it thecapability to cleave foreign sequences that may become mutated. Thisexcess energy model is also supported by previous studies demonstratingthat off-target effects can be reduced (but not eliminated) bydecreasing SpCas9 concentration (Hsu, P. D. et al. Nat Biotechnol 31,827-832 (2013); Pattanayak, V. et al. Nat Biotechnol 31, 839-843 (2013))or by reducing the complementarity length of the sgRNA (Fu, Y, et al.,Nat Biotechnol 32, 279-284 (2014), although other interpretations forthis effect have also been proposed (Josephs, E. A. et al. Nucleic AcidsRes 43, 8924-8941 (2015); Sternberg, S. H., et al. Nature 527, 110-113(2015); Kiani, S. et al. Nat Methods 12, 1051-1054 (2015))). Structuraldata suggests that the SpCas9-sgRNA-target DNA complex may be stabilizedby several SpCas9-mediated DNA contacts, including direct hydrogen bondsmade by four SpCas9 residues (N497, R661, Q695, Q926) to the phosphatebackbone of the target DNA strand (Nishimasu, H. et al. Cell 156,935-949 (2014); Anders, C., et al. Nature 513, 569-573 (2014)) (FIG. 1aand FIGS. 6a and 6b ). The present inventors envisioned that disruptionof one or more of these contacts might energetically poise theSpCas9-sgRNA complex at a level just sufficient to retain robuston-target activity but with a diminished ability to cleave mismatchedoff-target sites.

As described herein, Cas9 proteins can be engineered to show increasedspecificity, theoretically by reducing the binding affinity of Cas9 forDNA. Several variants of the widely used Streptococcus pyogenes Cas9(SpCas9) were engineered by introducing individual alanine substitutionsinto various residues in SpCas9 that might be expected to interact withphosphates on the DNA backbone using structural information, bacterialselection-based directed evolution, and combinatorial design. Thevariants were further tested for cellular activity using a robust E.coli-based screening assay to assess the cellular activities of thesevariants; in this bacterial system, cell survival depended on cleavageand subsequent destruction of a selection plasmid containing a gene forthe toxic gyrase poison ccdB and a 23 base pair sequence targeted by agRNA and SpCas9, and led to identification of residues that wereassociated with retained or lost activity. In addition, another SpCas9variant was identified and characterized, which exhibited improvedtarget specificity in human cells.

Furthermore, activities of single alanine substitution mutants of SpCas9as assessed in the bacterial cell-based system indicated that survivalpercentages between 50-100% usually indicated robust cleavage, whereas0% survival indicated that the enzyme had been functionally compromised.Additional mutations of SpCas9 were then assayed in bacteria to include:R63A, R66A, R69A, R70A, R71A, Y72A, R74A, R75A, K76A, N77A, R78A, R115A,H160A, K163A, R165A, L169A, R403A,T404A, F405A, N407A, R447A, N497A,I448A, Y450A, S460A, M495A, K510A, Y515A, R661A, M694A, Q695A, H698A,Y1013A, V1015A, R1122A, K1123A, K1124A, K1158A, K1185A, K1200A, S1216A,Q1221A, K1289A, R1298A, K1300A, K1325A, R1333A, K1334A, R1335A, andT1337A. With the exception of 2 mutants (R69A and F405A) that had <5%survival in bacteria, all of these additional single mutations appearedto have little effect on the on-target activity of SpCas9 (>70% survivalin the bacterial screen).

To further determine whether the variants of Cas9 identified in thebacterial screen functioned efficiently in human cells, various alaninesubstitution Cas9 mutants were tested using a human U2OS cell-basedEGFP-disruption assay. In this assay, successful cleavage of a targetsite in the coding sequence of a single integrated, constitutivelyexpressed EGFP gene led to the induction of indel mutations anddisruption of EGFP activity, which was quantitatively assessed by flowcytometry (see, for example, Reyon et al., Nat Biotechnol. 2012 May;30(5):460-5).

These experiments show that the results obtained in the bacterialcell-based assay correlate well with nuclease activities in human cells,suggesting that these engineering strategies could be extended to Cas9sfrom other species and different cells. Thus these findings providesupport for SpCas9 and SaCas9 variants, referred to collectively hereinas “variants” or “the variants”.

All of the variants described herein can be rapidly incorporated intoexisting and widely used vectors, e.g., by simple site-directedmutagenesis, and because they require only a small number of mutations,the variants should also work with other previously describedimprovements to the SpCas9 platform (e.g., truncated sgRNAs (Tsai etal., Nat Biotechnol 33, 187-197 (2015); Fu et al., Nat Biotechnol 32,279-284 (2014)), nickase mutations (Mali et al., Nat Biotechnol 31,833-838 (2013); Ran et al., Cell 154, 1380-1389 (2013)), FokI-dCas9fusions (Guilinger et al., Nat Biotechnol 32, 577-582 (2014); Tsai etal., Nat Biotechnol 32, 569-576 (2014); WO2014144288); and engineeredCRISPR-Cas9 nucleases with altered PAM specificities (Kleinstiver etal., Nature. 2015 Jul. 23; 523(7561):481-5).

Thus, provided herein are Cas9 variants, including SpCas9 variants. TheSpCas9 wild type sequence is as follows:

(SEQ ID NO: 1)         10         20         30         40MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR        50         60         70         80HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC        90        100        110        120YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG       130        140        150        160NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH       170        180        190        200MIKFRGHFLI EGDLNPDNSD VDKLFIQLVQ TYNQLFEENP       210        220        230        240INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN       250        260        270        280LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA       290        300        310        320QIGDQYADLF LAAKNLSDAI LLSDILRVNT EITKAPLSAS       330        340        350        360MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA       370        380        390        400GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR       410        420        430        440KQRTFDNGSI PHQIHLGELH AILRRQEDFY PFLKDNREKI       450        460        470        480EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE       490        500        510        520VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV       530        540        550        560YNELTKVKYV TEGMRKPAFL SGEQKKAIVD LLFKTNRKVT       570        580        590        600VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI       610        620        630        640IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA       650        660        670        680HLFDDKVMKQ LKRRRYTGWG RLSRKLINGI RDKQSGKTIL       690        700        710        720DFLKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL       730        740        750        760HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV       770        780        790        800IEMARENQTT QKGQKNSRER MKRIEEGIKE LGSQILKEHP       810        820        830        840VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDH       850        860        870        880IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK       890        900        910        920NYWRQLLNAK LITQRKFDNL TKAERGGLSE LDKAGFIKRQ       930        940        950        960LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS       970        980        990       1000KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK       1010       1020       1030       1040YPKLESEFVY GDYKVYDVRK MIAKSEQEIG KATAKYFFYS      1050       1060       1070       1080NIMNFFKTEI TLANGEIRKR PLIETNGETG EIVWDKGRDF      1090       1100       1110       1120ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI      1130       1140       1150       1160ARKKDWDPKK YGGFDSPTVA YSVLVVAKVE KGKSKKLKSV      1170       1180       1190       1200KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK      1210       1220       1230       1240YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS      1250       1260       1270       1280HYEKLKGSPE DNEQKQLFVE QHKHYLDEII EQISEFSKRV      1290       1300       1310       1320ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA      1330       1340       1350       1360 PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGD

The SpCas9 variants described herein can include the amino acid sequenceof SEQ ID NO:1, with mutations (i.e., replacement of the native aminoacid with a different amino acid, e.g., alanine, glycine, or serine), atone or more of the following positions: N497, R661, Q695, Q926 (or atpositions analogous thereto). In some embodiments, the SpCas9 variantsare at least 80%, e.g., at least 85%, 90%, or 95% identical to the aminoacid sequence of SEQ ID NO:1, e.g., have differences at up to 5%, 10%,15%, or 20% of the residues of SEQ ID NO:1 replaced, e.g., withconservative mutations, in addition to the mutations described herein.In preferred embodiments, the variant retains desired activity of theparent, e.g., the nuclease activity (except where the parent is anickase or a dead Cas9), and/or the ability to interact with a guide RNAand target DNA).

To determine the percent identity of two nucleic acid sequences, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). The length of a reference sequencealigned for comparison purposes is at least 80% of the length of thereference sequence, and in some embodiments is at least 90% or 100%. Thenucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein nucleic acid “identity” is equivalent to nucleic acid“homology”). The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.Percent identity between two polypeptides or nucleic acid sequences isdetermined in various ways that are within the skill in the art, forinstance, using publicly available computer software such as SmithWaterman Alignment (Smith, T. F. and M. S. Waterman (1981) J Mol Biol147:195-7); “BestFit” (Smith and Waterman, Advances in AppliedMathematics, 482-489 (1981)) as incorporated into GeneMatcher Plus™,Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure,Dayhof, M. O., Ed, pp 353-358; BLAST program (Basic Local AlignmentSearch Tool; (Altschul, S. F., W. Gish, et al. (1990) J Mol Biol 215:403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2,CLUSTAL, or Megalign (DNASTAR) software. In addition, those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thelength of the sequences being compared. In general, for proteins ornucleic acids, the length of comparison can be any length, up to andincluding full length (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, or 100%). For purposes of the present compositions andmethods, at least 80% of the full length of the sequence is aligned.

For purposes of the present invention, the comparison of sequences anddetermination of percent identity between two sequences can beaccomplished using a Blossum 62 scoring matrix with a gap penalty of 12,a gap extend penalty of 4, and a frameshift gap penalty of 5.

Conservative substitutions typically include substitutions within thefollowing groups: glycine, alanine; valine, isoleucine, leucine;aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine.

In some embodiments, the SpCas9 variants include one of the followingsets of mutations: N497A/R661A/Q695/Q926A (quadruple alanine mutant);Q695A/Q926A (double alanine mutant); R661A/Q695A/Q926A andN497A/Q695A/Q926A (triple alanine mutants). In some embodiments, theadditional substitution mutations at L169 and/or Y450 might be added tothese double-, triple, and quadruple mutants or added to single mutantsbearing substitutions at Q695 or Q926. In some embodiments, the mutantshave alanine in place of the wild type amino acid. In some embodiments,the mutants have any amino acid other than arginine or lysine (or thenative amino acid).

In some embodiments, the SpCas9 variants also include one of thefollowing mutations, which reduce or destroy the nuclease activity ofthe Cas9: D10, E762, D839, H983, or D986 and H840 or N863, e.g.,D10A/D10N and H840A/H840N/H840Y, to render the nuclease portion of theprotein catalytically inactive; substitutions at these positions couldbe alanine (as they are in Nishimasu al., Cell 156, 935-949 (2014)), orother residues, e.g., glutamine, asparagine, tyrosine, serine, oraspartate, e.g., E762Q, H983N, H983Y, D986N, N863D, N863S, or N863H (seeWO 2014/152432). In some embodiments, the variant includes mutations atD10A or H840A (which creates a single-strand nickase), or mutations atD10A and H840A (which abrogates nuclease activity; this mutant is knownas dead Cas9 or dCas9).

The SpCas9 N497A/R661A/Q695A/R926A mutations have analogous residues inStaphylococcus aureus Cas9 (SaCas9); see FIG. 14. Mutations to theresidues contacting the DNA or RNA backbone are expected to increase thespecificity of SaCas9 as we've observed for SpCas9. Thus, also providedherein are SaCas9 variants. The SaCas9 wild type sequence is as follows:

(SEQ ID NO: 2)         10         20         30         40MKRNYILGLD IGITSVGYGI IDYETRDVID AGVRLFKEAN         50        60         70         80VENNEGRRSK RGARRLKRRR RHRIQRVKKL LFDYNLLTDH        90        100        110        120SELSGINPYE ARVKGLSQKL SEEEFSAALL HLAKRRGVHN       130        140        150        160VNEVEEDTGN ELSTKEQISR NSKALEEKYV AELQLERLKK       170        180        190        200DGEVRGSINR FKTSDYVKEA KQLLKVQKAY HQLDQSFIDT       210        220        230        240YIDLLETRRT YYEGPGEGSP FGWKDIKEWY EMLMGHCTYF       250        260        270        280PEELRSVKYA YNADLYNALN DLNNLVITRD ENEKLEYYEK       290        300        310        320FQIIENVFKQ KKKPTLKQIA KEILVNEEDI KGYRVTSTGK       330        340        350        360PEFTNLKVYH DIKDITARKE IIENAELLDQ IAKILTIYQS       370        380        390        400SEDIQEELTN LNSELTQEEI EQISNLKGYT GTHNLSLKAI       410        420        430        440NLILDELWHT NDNQIAIFNR LKLVPKKVDL SQQKEIPTTL       450        460        470        480VDDFILSPVV KRSFIQSIKV INAIIKKYGL PNDIIIELAR       490        500        510        520EKNSKDAQKM INEMQKRNRQ TNERIEEIIR TTGKENAKYL       530        540        550        560IEKIKLHDMQ EGKCLYSLEA IPLEDLLNNP FNYEVDHIIP       570        580        590        600RSVSFDNSFN NKVLVKQEEN SKKGNRTPFQ YLSSSDSKIS       610        620        630        640YETFKKHILN LAKGKGRISK TKKEYLLEER DINRFSVQKD        650        660        670        680FINRNLVDTR YATRGLMNLL RSYFRVNNLD VKVKSINGGF       690        700        710        720TSFLRRKWKF KKERNKGYKH HAEDALIIAN ADFIFKEWKK       730        740        750        760LDKAKKVMEN QMFEEKQAES MPEIETEQEY KEIFITPHQI       770        780        790        800KHIKDFKDYK YSHRVDKKPN RELINDTLYS TRKDDKGNTL       810        820        830        840IVNNLNGLYD KDNDKLKKLI NKSPEKLLMY HHDPQTYQKL       850        860        870        880KLIMEQYGDE KNPLYKYYEE TGNYLTKYSK KDNGPVIKKI       890        900        910        920KYYGNKLNAH LDITDDYPNS RNKVVKLSLK PYRFDVYLDN       930        940        950        960GVYKFVTVKN LDVIKKENYY EVNSKCYEEA KKLKKISNQA       970        980        990       1000EFIASFYNND LIKINGELYR VIGVNNDLLN RIEVNMIDIT      1010       1020       1030       1040YREYLENMND KRPPRIIKTI ASKTQSIKKY STDILGNLYE       1050 VKSKKHPQII KKG

SaCas9 variants described herein include the amino acid sequence of SEQID NO:2, with mutations at one, two, three, four, five, or all six ofthe following positions: Y211, W229, R245, T392, N419, and/or R654,e.g., comprising a sequence that is at least 80% identical to the aminoacid sequence of SEQ ID NO:2 with mutations at one, two, three, fourfive or six of the following positions: Y211, W229, R245, T392, N419,and/or R654.

In some embodiments, the variant SaCas9 proteins also comprise one ormore of the following mutations: Y211A; W229A; Y230A; R245A; T392A;N419A; L446A; Y651A; R654A; D786A; T787A; Y789A; T882A; K886A; N888A;A889A; L909A; N985A; N986A; R991A; R1015A; N44A; R45A; R51A; R55A; R59A;R60A; R116A; R165A; N169A; R208A; R209A; Y211A; T238A; Y239A; K248A;Y256A; R314A; N394A; Q414A; K57A; R61A; H111A; K114A; V164A; R165A;L788A; S790A; R792A; N804A; Y868A; K870A; K878A; K879A; K881A; Y897A;R901A; K906A.

In some embodiments, variant SaCas9 proteins comprise one or more of thefollowing additional mutations: Y211A, W229A, Y230A, R245A, T392A,N419A, L446A, Y651A, R654A, D786A, T787A, Y789A, T882A, K886A, N888A,A889A, L909A, N985A, N986A, R991A, R1015A, N44A, R45A, R51A, R55A, R59A,R60A, R116A, R165A, N169A, R208A, R209A, Y211A, T238A, Y239A, K248A,Y256A, R314A, N394A, Q414A, K57A, R61A, H111A, K114A, V164A, R165A,L788A, S790A, R792A, N804A, Y868A, K870A, K878A, K879A, K881A, Y897A,R901A, K906A.

In some embodiments, the variant SaCas9 proteins comprise multiplesubstitution mutations: R245/T392/N419/R654 and Y221/R245/N419/R654(quadruple variant mutants); N419/R654, R245/R654, Y221/R654, andY221/N419 (double mutants); R245/N419/R654, Y211/N419/R654, andT392/N419/R654 (triple mutants). In some embodiments the mutants containalanine in place of the wild type amino acid.

In some embodiments, the variant SaCas9 proteins also comprise mutationsat E782K, K929R, N968K, and/or R1015H. For example, the KKH variant(E782K/N968K/R1015H), the KRH variant (E782K/K929R/R1015H), or the KRKHvariant (E782K/K929R/N968K/R1015H)]

In some embodiments, the variant SaCas9 proteins also comprise one ormore mutations that decrease nuclease activity selected from the groupconsisting of mutations at D10, E477, D556, H701, or D704; and at H557or N580.

In some embodiments, the mutations are: (i) D10A or D10N, (ii) H557A,H557N, or H557Y, (iii) N580A, and/or (iv) D556A.

Also provided herein are isolated nucleic acids encoding the Cas9variants, vectors comprising the isolated nucleic acids, optionallyoperably linked to one or more regulatory domains for expressing thevariant proteins, and host cells, e.g., mammalian host cells, comprisingthe nucleic acids, and optionally expressing the variant proteins.

The variants described herein can be used for altering the genome of acell; the methods generally include expressing the variant proteins inthe cells, along with a guide RNA having a region complementary to aselected portion of the genome of the cell. Methods for selectivelyaltering the genome of a cell are known in the art, see, e.g., U.S. Pat.No. 8,993,233; US 20140186958; U.S. Pat. No. 9,023,649; WO/2014/099744;WO 2014/089290; WO2014/144592; WO144288; WO2014/204578; WO2014/152432;WO2115/099850; U.S. Pat. No. 8,697,359; US20160024529; US20160024524;US20160024523; US20160024510; US20160017366; US20160017301;US20150376652; US20150356239; US20150315576; US20150291965;US20150252358; US20150247150; US20150232883; US20150232882;US20150203872; US20150191744; US20150184139; US20150176064;US20150167000; US20150166969; US20150159175; US20150159174;US20150093473; US20150079681; US20150067922; US20150056629;US20150044772; US20150024500; US20150024499; US20150020223;US20140356867; US20140295557; US20140273235; US20140273226;US20140273037; US20140189896; US20140113376; US20140093941;US20130330778; US20130288251; US20120088676; US20110300538;US20110236530; US20110217739; US20110002889; US20100076057;US20110189776; US20110223638; US20130130248; US20150050699;US20150071899; US20150045546; US20150031134; US20150024500;US20140377868; US20140357530; US20140349400; US20140335620;US20140335063; US20140315985; US20140310830; US20140310828;US20140309487; US20140304853; US20140298547; US20140295556;US20140294773; US20140287938; US20140273234; US20140273232;US20140273231; US20140273230; US20140271987; US20140256046;US20140248702; US20140242702; US20140242700; US20140242699;US20140242664; US20140234972; US20140227787; US20140212869;US20140201857; US20140199767; US20140189896; US20140186958;US20140186919; US20140186843; US20140179770; US20140179006;US20140170753; WO/2008/108989; WO/2010/054108; WO/2012/164565;WO/2013/098244; WO/2013/176772; Makarova et al., “Evolution andclassification of the CRISPR-Cas systems” 9(6) Nature ReviewsMicrobiology 467-477 (1-23) (June 2011); Wiedenheft et al., “RNA-guidedgenetic silencing systems in bacteria and archaea” 482 Nature 331-338(Feb. 16, 2012); Gasiunas et al., “Cas9-crRNA ribonucleoprotein complexmediates specific DNA cleavage for adaptive immunity in bacteria”109(39) Proceedings of the National Academy of Sciences USA E2579-E2586(Sep. 4, 2012); Jinek et al., “A Programmable Dual-RNA-Guided DNAEndonuclease in Adaptive Bacterial Immunity” 337 Science 816-821 (Aug.17, 2012); Carroll, “A CRISPR Approach to Gene Targeting” 20(9)Molecular Therapy 1658-1660 (September 2012); U.S. Appl. No. 61/652,086,filed May 25, 2012; Al-Attar et al., Clustered Regularly InterspacedShort Palindromic Repeats (CRISPRs): The Hallmark of an IngeniousAntiviral Defense Mechanism in Prokaryotes, Biol Chem. (2011) vol. 392,Issue 4, pp. 277-289; Hale et al., Essential Features and RationalDesign of CRISPR RNAs That Function With the Cas RAMP Module Complex toCleave RNAs, Molecular Cell, (2012) vol. 45, Issue 3, 292-302.

The variant proteins described herein can be used in place of or inaddition to any of the Cas9 proteins described in the foregoingreferences, or in combination with mutations described therein. Inaddition, the variants described herein can be used in fusion proteinsin place of the wild-type Cas9 or other Cas9 mutations (such as thedCas9 or Cas9 nickase described above) as known in the art, e.g., afusion protein with a heterologous functional domains as described inU.S. Pat. No. 8,993,233; US 20140186958; U.S. Pat. No. 9,023,649;WO/2014/099744; WO 2014/089290; WO2014/144592; WO144288; WO2014/204578;WO2014/152432; WO2115/099850; U.S. Pat. No. 8,697,359; US2010/0076057;US2011/0189776; US2011/0223638; US2013/0130248; WO/2008/108989;WO/2010/054108; WO/2012/164565; WO/2013/098244; WO/2013/176772;US20150050699; US 20150071899 and WO 2014/124284. For example, thevariants, preferably comprising one or more nuclease-reducing or killingmutation, can be fused on the N or C terminus of the Cas9 to atranscriptional activation domain or other heterologous functionaldomains (e.g., transcriptional repressors (e.g., KRAB, ERD, SID, andothers, e.g., amino acids 473-530 of the ets2 repressor factor (ERF)repressor domain (ERD), amino acids 1-97 of the KRAB domain of KOX1, oramino acids 1-36 of the Mad mSIN3 interaction domain (SID); see Beerliet al., PNAS USA 95:14628-14633 (1998)) or silencers such asHeterochromatin Protein 1 (HP1, also known as swi6), e.g., HP1α or HP1β;proteins or peptides that could recruit long non-coding RNAs (lncRNAs)fused to a fixed RNA binding sequence such as those bound by the MS2coat protein, endoribonuclease Csy4, or the lambda N protein; enzymesthat modify the methylation state of DNA (e.g., DNA methyltransferase(DNMT) or TET proteins); or enzymes that modify histone subunits (e.g.,histone acetyltransferases (HAT), histone deacetylases (HDAC), histonemethyltransferases (e.g., for methylation of lysine or arginineresidues) or histone demethylases (e.g., for demethylation of lysine orarginine residues)) as are known in the art can also be used. A numberof sequences for such domains are known in the art, e.g., a domain thatcatalyzes hydroxylation of methylated cytosines in DNA. Exemplaryproteins include the Ten-Eleven-Translocation (TET)1-3 family, enzymesthat converts 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC)in DNA.

Sequences for human TET1-3 are known in the art and are shown in thefollowing table:

GenBank Accession Nos. Gene Amino Acid Nucleic Acid TET1 NP_085128.2NM_030625.2 TET2* NP_001120680.1 (var 1) NM_001127208.2 NP_060098.3 (var2) NM_017628.4 TET3 NP_659430.1 NM_144993.1 *Variant (1) represents thelonger transcript and encodes the longer isoform (a). Variant (2)differs in the 5′ UTR and in the 3′ UTR and coding sequence compared tovariant 1. The resulting isoform (b) is shorter and has a distinctC-terminus compared to isoform a.

In some embodiments, all or part of the full-length sequence of thecatalytic domain can be included, e.g., a catalytic module comprisingthe cysteine-rich extension and the 2OGFeDO domain encoded by 7 highlyconserved exons, e.g., the Tet1 catalytic domain comprising amino acids1580-2052, Tet2 comprising amino acids 1290-1905 and Tet3 comprisingamino acids 966-1678. See, e.g., FIG. 1 of Iyer et al., Cell Cycle. 2009Jun. 1; 8(11):1698-710. Epub 2009 Jun. 27, for an alignment illustratingthe key catalytic residues in all three Tet proteins, and thesupplementary materials thereof (available at ftp siteftp.ncbi.nih.gov/pub/aravind/DONS/supplementary_material_DONS.html) forfull length sequences (see, e.g., seq 2c); in some embodiments, thesequence includes amino acids 1418-2136 of Tet1 or the correspondingregion in Tet2/3.

Other catalytic modules can be from the proteins identified in Iyer etal., 2009.

In some embodiments, the heterologous functional domain is a biologicaltether, and comprises all or part of (e.g., DNA binding domain from) theMS2 coat protein, endoribonuclease Csy4, or the lambda N protein. Theseproteins can be used to recruit RNA molecules containing a specificstem-loop structure to a locale specified by the dCas9 gRNA targetingsequences. For example, a dCas9 variant fused to MS2 coat protein,endoribonuclease Csy4, or lambda N can be used to recruit a longnon-coding RNA (lncRNA) such as XIST or HOTAIR; see, e.g., Keryer-Bibenset al., Biol. Cell 100:125-138 (2008), that is linked to the Csy4, MS2or lambda N binding sequence. Alternatively, the Csy4, MS2 or lambda Nprotein binding sequence can be linked to another protein, e.g., asdescribed in Keryer-Bibens et al., supra, and the protein can betargeted to the dCas9 variant binding site using the methods andcompositions described herein. In some embodiments, the Csy4 iscatalytically inactive. In some embodiments, the Cas9 variant,preferably a dCas9 variant, is fused to FokI as described in U.S. Pat.No. 8,993,233; US 20140186958; U.S. Pat. No. 9,023,649; WO/2014/099744;WO 2014/089290; WO2014/144592; WO144288; WO2014/204578; WO2014/152432;WO2115/099850; U.S. Pat. No. 8,697,359; US2010/0076057; US2011/0189776;US2011/0223638; US2013/0130248; WO/2008/108989; WO/2010/054108;WO/2012/164565; WO/2013/098244; WO/2013/176772; US20150050699; US20150071899 and WO 2014/204578.

In some embodiments, the fusion proteins include a linker between thedCas9 variant and the heterologous functional domains. Linkers that canbe used in these fusion proteins (or between fusion proteins in aconcatenated structure) can include any sequence that does not interferewith the function of the fusion proteins. In preferred embodiments, thelinkers are short, e.g., 2-20 amino acids, and are typically flexible(i.e., comprising amino acids with a high degree of freedom such asglycine, alanine, and serine). In some embodiments, the linker comprisesone or more units consisting of GGGS (SEQ ID NO:3) or GGGGS (SEQ IDNO:4), e.g., two, three, four, or more repeats of the GGGS (SEQ ID NO:5)or GGGGS (SEQ ID NO:6) unit. Other linker sequences can also be used.

In some embodiments, the variant protein includes a cell-penetratingpeptide sequence that facilitates delivery to the intracellular space,e.g., HIV-derived TAT peptide, penetratins, transportans, or hCT derivedcell-penetrating peptides, see, e.g., Caron et al., (2001) Mol Ther.3(3):310-8; Langel, Cell-Penetrating Peptides: Processes andApplications (CRC Press, Boca Raton Fla. 2002); El-Andaloussi et al.,(2005) Curr Pharm Des. 11(28):3597-611; and Deshayes et al., (2005) CellMol Life Sci. 62(16):1839-49.

Cell penetrating peptides (CPPs) are short peptides that facilitate themovement of a wide range of biomolecules across the cell membrane intothe cytoplasm or other organelles, e.g. the mitochondria and thenucleus. Examples of molecules that can be delivered by CPPs includetherapeutic drugs, plasmid DNA, oligonucleotides, siRNA, peptide-nucleicacid (PNA), proteins, peptides, nanoparticles, and liposomes. CPPs aregenerally 30 amino acids or less, are derived from naturally ornon-naturally occurring protein or chimeric sequences, and containeither a high relative abundance of positively charged amino acids, e.g.lysine or arginine, or an alternating pattern of polar and non-polaramino acids. CPPs that are commonly used in the art include Tat (Frankelet al., (1988) Cell. 55:1189-1193, Vives et al., (1997) J. Biol. Chem.272:16010-16017), penetratin (Derossi et al., (1994) J. Biol. Chem.269:10444-10450), polyarginine peptide sequences (Wender et al., (2000)Proc. Natl. Acad. Sci. USA 97:13003-13008, Futaki et al., (2001) J.Biol. Chem. 276:5836-5840), and transportan (Pooga et al., (1998) Nat.Biotechnol. 16:857-861).

CPPs can be linked with their cargo through covalent or non-covalentstrategies. Methods for covalently joining a CPP and its cargo are knownin the art, e.g. chemical cross-linking (Stetsenko et al., (2000) J.Org. Chem. 65:4900-4909, Gait et al. (2003) Cell. Mol. Life. Sci.60:844-853) or cloning a fusion protein (Nagahara et al., (1998) Nat.Med. 4:1449-1453). Non-covalent coupling between the cargo and shortamphipathic CPPs comprising polar and non-polar domains is establishedthrough electrostatic and hydrophobic interactions.

CPPs have been utilized in the art to deliver potentially therapeuticbiomolecules into cells. Examples include cyclosporine linked topolyarginine for immunosuppression (Rothbard et al., (2000) NatureMedicine 6(11):1253-1257), siRNA against cyclin B1 linked to a CPPcalled MPG for inhibiting tumorigenesis (Crombez et al., (2007) BiochemSoc. Trans. 35:44-46), tumor suppressor p53 peptides linked to CPPs toreduce cancer cell growth (Takenobu et al., (2002) Mol. Cancer Ther.1(12):1043-1049, Snyder et al., (2004) PLoS Biol. 2:E36), and dominantto negative forms of Ras or phosphoinositol 3 kinase (PI3K) fused to Tatto treat asthma (Myou et al., (2003) J. Immunol. 171:4399-4405).

CPPs have been utilized in the art to transport contrast agents intocells for imaging and biosensing applications. For example, greenfluorescent protein (GFP) attached to Tat has been used to label cancercells (Shokolenko et al., (2005) DNA Repair 4(4):511-518). Tatconjugated to quantum dots have been used to successfully cross theblood-brain barrier for visualization of the rat brain (Santra et al.,(2005) Chem. Commun. 3144-3146). CPPs have also been combined withmagnetic resonance imaging techniques for cell imaging (Liu et al.,(2006) Biochem. and Biophys. Res. Comm. 347(1):133-140). See also Ramseyand Flynn, Pharmacol Ther. 2015 Jul. 22. pii: 50163-7258(15)00141-2.

Alternatively or in addition, the variant proteins can include a nuclearlocalization sequence, e.g., SV40 large T antigen NLS (PKKKRRV (SEQ IDNO:7)) and nucleoplasmin NLS (KRPAATKKAGQAKKKK (SEQ ID NO:8)). OtherNLSs are known in the art; see, e.g., Cokol et al., EMBO Rep. 2000 Nov.15; 1(5): 411-415; Freitas and Cunha, Curr Genomics. 2009 December;10(8): 550-557.

In some embodiments, the variants include a moiety that has a highaffinity for a ligand, for example GST, FLAG or hexahistidine sequences.Such affinity tags can facilitate the purification of recombinantvariant proteins.

For methods in which the variant proteins are delivered to cells, theproteins can be produced using any method known in the art, e.g., by invitro translation, or expression in a suitable host cell from nucleicacid encoding the variant protein; a number of methods are known in theart for producing proteins. For example, the proteins can be produced inand purified from yeast, E. coli, insect cell lines, plants, transgenicanimals, or cultured mammalian cells; see, e.g., Palomares et al.,“Production of Recombinant Proteins: Challenges and Solutions,” MethodsMol Biol. 2004; 267:15-52. In addition, the variant proteins can belinked to a moiety that facilitates transfer into a cell, e.g., a lipidnanoparticle, optionally with a linker that is cleaved once the proteinis inside the cell. See, e.g., LaFountaine et al., Int J Pharm. 2015Aug. 13; 494(1):180-194.

Expression Systems

To use the Cas9 variants described herein, it may be desirable toexpress them from a nucleic acid that encodes them. This can beperformed in a variety of ways. For example, the nucleic acid encodingthe Cas9 variant can be cloned into an intermediate vector fortransformation into prokaryotic or eukaryotic cells for replicationand/or expression. Intermediate vectors are typically prokaryotevectors, e.g., plasmids, or shuttle vectors, or insect vectors, forstorage or manipulation of the nucleic acid encoding the Cas9 variantfor production of the Cas9 variant. The nucleic acid encoding the Cas9variant can also be cloned into an expression vector, for administrationto a plant cell, animal cell, preferably a mammalian cell or a humancell, fungal cell, bacterial cell, or protozoan cell.

To obtain expression, a sequence encoding a Cas9 variant is typicallysubcloned into an expression vector that contains a promoter to directtranscription. Suitable bacterial and eukaryotic promoters are wellknown in the art and described, e.g., in Sambrook et al., MolecularCloning, A Laboratory Manual (3d ed. 2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 2010). Bacterial expressionsystems for expressing the engineered protein are available in, e.g., E.coli, Bacillus sp., and Salmonella (Palva et al., 1983, Gene22:229-235). Kits for such expression systems are commerciallyavailable. Eukaryotic expression systems for mammalian cells, yeast, andinsect cells are well known in the art and are also commerciallyavailable.

The promoter used to direct expression of a nucleic acid depends on theparticular application. For example, a strong constitutive promoter istypically used for expression and purification of fusion proteins. Incontrast, when the Cas9 variant is to be administered in vivo for generegulation, either a constitutive or an inducible promoter can be used,depending on the particular use of the Cas9 variant. In addition, apreferred promoter for administration of the Cas9 variant can be a weakpromoter, such as HSV TK or a promoter having similar activity. Thepromoter can also include elements that are responsive totransactivation, e.g., hypoxia response elements, Gal4 responseelements, lac repressor response element, and small molecule controlsystems such as tetracycline-regulated systems and the RU-486 system(see, e.g., Gossen & Bujard, 1992, Proc. Natl. Acad. Sci. USA, 89:5547;Oligino et al., 1998, Gene Ther., 5:491-496; Wang et al., 1997, GeneTher., 4:432-441; Neering et al., 1996, Blood, 88:1147-55; and Rendahlet al., 1998, Nat. Biotechnol., 16:757-761).

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the nucleic acid inhost cells, either prokaryotic or eukaryotic. A typical expressioncassette thus contains a promoter operably linked, e.g., to the nucleicacid sequence encoding the Cas9 variant, and any signals required, e.g.,for efficient polyadenylation of the transcript, transcriptionaltermination, ribosome binding sites, or translation termination.Additional elements of the cassette may include, e.g., enhancers, andheterologous spliced intronic signals.

The particular expression vector used to transport the geneticinformation into the cell is selected with regard to the intended use ofthe Cas9 variant, e.g., expression in plants, animals, bacteria, fungus,protozoa, etc. Standard bacterial expression vectors include plasmidssuch as pBR322 based plasmids, pSKF, pET23D, and commercially availabletag-fusion expression systems such as GST and LacZ.

Expression vectors containing regulatory elements from eukaryoticviruses are often used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+,pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 late promoter, metallothionein promoter, murine mammary tumor viruspromoter, Rous sarcoma virus promoter, polyhedrin promoter, or otherpromoters shown effective for expression in eukaryotic cells.

The vectors for expressing the Cas9 variants can include RNA Pol IIIpromoters to drive expression of the guide RNAs, e.g., the H1, U6 or 7SKpromoters. These human promoters allow for expression of Cas9 variantsin mammalian cells following plasmid transfection.

Some expression systems have markers for selection of stably transfectedcell lines such as thymidine kinase, hygromycin B phosphotransferase,and dihydrofolate reductase. High yield expression systems are alsosuitable, such as using a baculovirus vector in insect cells, with thegRNA encoding sequence under the direction of the polyhedrin promoter orother strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of recombinant sequences.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of protein,which are then purified using standard techniques (see, e.g., Colley etal., 1989, J. Biol. Chem., 264:17619-22; Guide to Protein Purification,in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)).Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, 1977, J.Bacteriol. 132:349-351; Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Any of the known procedures for introducing foreign nucleotide sequencesinto host cells may be used. These include the use of calcium phosphatetransfection, polybrene, protoplast fusion, electroporation,nucleofection, liposomes, microinjection, naked DNA, plasmid vectors,viral vectors, both episomal and integrative, and any of the otherwell-known methods for introducing cloned genomic DNA, cDNA, syntheticDNA or other foreign genetic material into a host cell (see, e.g.,Sambrook et al., supra). It is only necessary that the particulargenetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingthe Cas9 variant.

The present invention also includes the vectors and cells comprising thevectors.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Methods

Bacterial-Based Positive Selection Assay for Evolving SpCas9 Variants

Competent E. coli BW25141(λDE3)²³ containing a positive selectionplasmid (with embedded target site) were transformed withCas9/sgRNA-encoding plasmids. Following a 60 minute recovery in SOBmedia, transformations were plated on LB plates containing eitherchloramphenicol (non-selective) or chloramphenicol+10 mM arabinose(selective).

To identify additional positions that might be critical for genome widetarget specificity, a bacterial selection system previously used tostudy properties of homing endonucleases (hereafter referred to as thepositive selection) (Chen & Zhao, Nucleic Acids Res 33, e154 (2005);Doyon et al., J Am Chem Soc 128, 2477-2484 (2006)) was adapted.

In the present adaptation of this system, Cas9-mediated cleavage of apositive selection plasmid encoding an inducible toxic gene enables cellsurvival, due to subsequent degradation and loss of the linearizedplasmid. After establishing that SpCas9 can function in the positiveselection system, both wild-type and the variants were tested for theirability to cleave a selection plasmid harboring a target site selectedfrom the known human genome. These variants were introduced intobacteria with a positive selection plasmid containing a target site andplated on selective medium. Cleavage of the positive selection plasmidwas estimated by calculating the survival frequency: colonies onselective plates/colonies on non-selective plates (see FIG. 1, 5-6).

A subset of plasmids used in this study (sequences shown below) AddgeneName ID Description JDS246 43861 CMV-T7-humanSpCas9-NLS-3xFLAG VP12pending CMV-T7-humanSpCas9-HF1(N497A, R661A, Q695A, Q926A)-NLS-3xFLAGMSP2135 pending CMV-T7-humanSpCas9-HF2(N497A, R661A, Q695A, Q926A,D1135E)-NLS-3xFLAG MSP2133 pending CMV-T7-humanSpCas9-HF4(Y450A, N497A,R661A, Q695A, Q926A)-NLS-3xFLAG MSP469 65771CMV-T7-humanSpCas9-VQR(D1135V, R1335Q, T1337R)-NLS-3xFLAG MSP2440pending CMV-T7-humanSpCas9-VQR-HF1(N497A, R661A, Q695A, Q926A, D1135V,R1335Q, T1337R)-NLS-3xFLAG BPK2797 pendingCMV-T7-humanSpCas9-VRQR(D1135V, G1218R, R1335Q, T1337R)-NLS-3xFLAGMSP2443 pending CMV-T7-humanSpCas9-VRQR-HF1(N497A, R661A, Q695A, Q926A,D1135V, G1218R, R1335Q, T1337R)-NLS-3xFLAG BPK1520 65777U6-BsmBlcassette-Sp-sgRNA

Human Cell Culture and Transfection

U2OS.EGFP cells harboring a single integrated copy of a constitutivelyexpressed EGFP-PEST reporter gene¹⁵ were cultured in Advanced DMEM media(Life Technologies) supplemented with 10% FBS, 2 mM GlutaMax (LifeTechnologies), penicillin/streptomycin, and 400 μg/ml of G418 at 37° C.with 5% CO₂. Cells were co-transfected with 750 ng of Cas9 plasmid and250 ng of sgRNA plasmid (unless otherwise noted) using the DN-100program of a Lonza 4D-nucleofector according to the manufacturer'sprotocols. Cas9 plasmid transfected together with an empty U6 promoterplasmid was used as a negative control for all human cell experiments.(see FIGS. 2, 7-10).

Human Cell EGFP Disruption Assay

EGFP disruption experiments were performed as previously described¹⁶.Transfected cells were analyzed for EGFP expression ˜52 hourspost-transfection using a Fortessa flow cytometer (BD Biosciences).Background EGFP loss was gated at approximately 2.5% for all experiments(see FIGS. 2, 7).

T7E1 Assay, Targeted Deep-Sequencing, and GUIDE-Seq to QuantifyNuclease-Induced Mutation Rates

T7E1 assays were performed as previously described for human cells(Kleinstiver, B. P. et al., Nature 523, 481-485 (2015)). For U2OS.EGFPhuman cells, genomic DNA was extracted from transfected cells ˜72 hourspost-transfection using the Agencourt DNAdvance Genomic DNA IsolationKit (Beckman Coulter Genomics). Roughly 200 ng of purified PCR productwas denatured, annealed, and digested with T7E1 (New England BioLabs).Mutagenesis frequencies were quantified using a Qiaxcel capillaryelectrophoresis instrument (Qlagen), as previously described for humancells¹⁵.

GUIDE-seq experiments were performed as previously described (Tsai etal., Nat Biotechnol 33, 187-197 (2015)). Briefly, phosphorylated,phosphorothioate-modified double-stranded oligodeoxynucleotides (dsODNs)were transfected into U2OS cells with Cas9 nuclease along with Cas9 andsgRNA expression plasmids, as described above. dsODN-specificamplification, high-throughput sequencing, and mapping were performed toidentify genomic intervals containing DSB activity. For wild-type versusdouble or quadruple mutant variant experiments, off-target read countswere normalized to the on-target read counts to correct for sequencingdepth differences between samples. The normalized ratios for wild-typeand variant SpCas9 were then compared to calculate the fold-change inactivity at off-target sites. To determine whether wild-type and SpCas9variant samples for GUIDE-seq had similar oligo tag integration rates atthe intended target site, restriction fragment length polymorphism(RFLP) assays were performed by amplifying the intended target loci withPhusion Hot-Start Flex from 100 ng of genomic DNA (isolated as describedabove). Roughly 150 ng of PCR product was digested with 20 U of NdeI(New England BioLabs) for 3 hours at 37° C. prior to clean-up using theAgencourt Ampure XP kit. RFLP results were quantified using a Qiaxcelcapillary electrophoresis instrument (Qlagen) to approximate oligo tagintegration rates. T7E1 assays were performed for a similar purpose, asdescribed above.

Example 1

One potential solution to address targeting specificity of CRISPR-Cas9RNA guided gene editing would be to engineer Cas9 variants with novelmutations.

Based on these earlier results, it was hypothesized (without wishing tobe bound by theory) that the specificity of CRISPR-Cas9 nucleases mightbe significantly increased by reducing the non-specific binding affinityof Cas9 for DNA, mediated by the binding to the phosphate groups on theDNA or hydrophobic or base stacking interactions with the DNA. Thisapproach would have the advantage of not decreasing the length of thetarget site recognized by the gRNA/Cas9 complex, as in the previouslydescribed truncated gRNA approach. It was reasoned that non-specificbinding affinity of Cas9 for DNA might be reduced by mutating amino acidresidues that contact phosphate groups on the target DNA.

An analogous approach has been used to create variants of non-Cas9nucleases such as TALENs (see, for example, Guilinger et al., Nat.Methods. 11: 429 (2014)).

In an initial test of the hypothesis, the present inventors attempted toengineer a reduced affinity variant of the widely used S. pyogenes Cas9(SpCas9) by introducing individual alanine substitutions into variousresidues in SpCas9 that might be expected to interact with phosphates onthe DNA backbone. An E. coli-based screening assay was used to assessthe activities of these variants (Kleinstiver et al., Nature. 2015 Jul.23; 523(7561):481-5). In this bacterial system, cell survival dependedon cleavage (and subsequent destruction) of a selection plasmidcontaining a gene for the toxic gyrase poison ccdB and a 23 base pairsequence targeted by a gRNA and SpCas9. Results of this experimentidentified residues that retained or lost activity (Table 1).

TABLE 1 Activities of single alanine substitution mutants of Cas9 asassessed in the bacterial cell-based system shown in FIG. 1. mutation %survival R63A 84.2 R66A 0 R70A 0 R74A 0 R78A 56.4 R165A 68.9 R403A 85.2N407A 97.2 N497A 72.6 K510A 79.0 Y515A 34.1 R661A 75.0 Q695A 69.8 Q926A53.3 K1107A 47.4 E1108A 40.0 S1109A 96.6 K1113A 51.8 R1114A 47.3 S1116A73.8 K1118A 48.7 D1135A 67.2 S1136A 69.2 K1151A 0 K1153A 76.6 K1155A44.6 K1158A 46.5 K1185A 19.3 K1200A 24.5 S1216A 100.4 Q1221A 98.8 K1289A55.2 R1298A 28.6 K1300A 59.8 K1325A 52.3 R1333A 0 K1334A 87.5 R1335A 0T1337A 64.6Survival percentages between 50-100% usually indicated robust cleavage,whereas 0% survival indicated that the enzyme has been functionallycompromised. Additional mutations that were assayed in bacteria (but arenot shown in the table above) include: R69A, R71A, Y72A, R75A, K76A,N77A, R115A, H160A, K163A, L169A, T404A, F405A, R447A, 1448A, Y450A,S460A, M495A, M694A, H698A, Y1013A, V1015A, R1122A, K1123A, and K1124A.With the exception of R69A and F405A (which had <5% survival inbacteria), all of these additional single mutations appeared to havelittle effect on the on-target activity of SpCas9 (>70% survival in thebacterial screen).

15 different SpCas9 variants bearing all possible single, double, tripleand quadruple combinations of the N497A, R661A, Q695A, and Q926Amutations were constructed to test whether contacts made by theseresidues might be dispensable for on-target activity (FIG. 1b ). Forthese experiments, a previously described human cell-based assay wasused in which cleavage and induction of insertion or deletion mutations(indels) by non-homologous end-joining (NHEJ)-mediated repair within asingle integrated EGFP reporter gene leads to loss of cell fluorescence(Reyon, D. et al., Nat Biotechnol. 30, 460-465, 2012). Using aEGFP-targeted sgRNA previously shown to efficiently disrupt EGFPexpression in human cells when paired with wild-type SpCas9 (Fu, Y. etal., Nat Biotechnol 31, 822-826 (2013), all 15 SpCas9 variants possessedEGFP disruption activities comparable to that of wild-type SpCas9 (FIG.1b , grey bars). Thus, substitution of one or all of these residues didnot reduce on-target cleavage efficiency of SpCas9 with thisEGFP-targeted sgRNA.

Next, experiments were performed to assess the relative activities ofall 15 SpCas9 variants at mismatched target sites. To do this, the EGFPdisruption assay was repeated with derivatives of the EGFP-targetedsgRNA used in the previous experiment that contain pairs of substitutedbases at positions 13 and 14, 15 and 16, 17 and 18, and 18 and 19(numbering starting with 1 for the most PAM-proximal base and endingwith 20 for the most PAM-distal base; FIG. 1b ). This analysis revealedthat one of the triple mutants (R661A/Q695A/Q926A) and the quadruplemutant (N497A/R661A/Q695A/Q926A) both showed levels of EGFP disruptionequivalent to that of background with all four of the mismatched sgRNAs(FIG. 1b , colored bars). Notably, among the 15 variants, thosepossessing the lowest activities with the mismatched sgRNAs all harboredthe Q695A and Q926A mutations. Based on these results and similar datafrom an experiment using a sgRNA for another EGFP target site, thequadruple mutant (N497A/R661A/Q695A/Q926A) was chosen for additionalanalysis and designated it as SpCas9-HF1 (for high-fidelity variant #1).

On-Target Activities of SpCas9-HF1

To determine how robustly SpCas9-HF1 functions at a larger number ofon-target sites, direct comparisons were performed between this variantand wild-type SpCas9 using additional sgRNAs. In total, 37 differentsgRNAs were tested: 24 targeted to EGFP (assayed with the EGFPdisruption assay) and 13 targeted to endogenous human gene targets(assayed using the T7 Endonuclease I (T7EI) mismatch assay). 20 of the24 sgRNAs tested with the EGFP disruption assay (FIG. 1c ) and 12 of the13 sgRNAs tested on endogenous human gene sites (FIG. 1d ) showedactivities with SpCas9-HF1 that were at least 70% as active as wild-typeSpCas9 with the same sgRNA (FIG. 1e ). Indeed, SpCas9-HF1 showed highlycomparable activities (90-140%) to wild-type SpCas9 with the vastmajority of sgRNAs (FIG. 1e ). Three of the 37 sgRNAs tested showedessentially no activity with SpCas9-HF1 and examination of these targetsites did not suggest any obvious differences in the characteristics ofthese sequences compared to those for which high activities were seen(Table 3). Overall, SpCas9-HF1 possessed comparable activities (greaterthan 70% of wild-type SpCas9 activities) for 86% (32/37) of the sgRNAstested.

TABLE 3 List of sgRNA targets EGFP Spacer SEQ Prep length ID Sequencewith extended SEQ ID Name Name (nt) Spacer Sequence NO: PAM NO: S.pyogenes sgRNAs FYF1 NGG 20 GGGCACGGGC 9. GGGCACGGGCAGCTTGC 10. 320 site1 AGCTTGCCGG CGGTGGT FYF1 NGG 18 GCACGGGCAG 11. GCACGGGCAGCTTGCCG 12.641 site 1 CTTGCCGG GTGGT CK10 NGG 20 GGGCACccGCA 13. GGGCACccGCAGCTTGC14. 12 site 1- GCTTGCCGG CGGTGGT 13 & 14 FYF1 NGG 20 GGGCtgGGGCA 15.GGGCtgGGGCAGCTTGC 16. 429 site 1- GCTTGCCGG CGGTGGT 15 & 16 FYF1 NGG 20GGcgACGGGCA 17. GGcgACGGGCAGCTTGC 18. 430 site 1- GCTTGCCGG CGGTGGT 17 &18 FYF1 NGG 20 GccCACGGGCA 19. GccCACGGGCAGCTTGC 20. 347 site 1-GCTTGCCGG CGGTGGT 18 & 19 BPK1 NGG 20 GTCGCCCTCG 21. GTCGCCCTCGAACTTCA22. 345 site 2 AACTTCACCT CCTCGGC BPK1 NGG 20 GTAGGTCAGG 23.GTAGGTCAGGGTGGTCA 24. 350 site 3 GTGGTCACGA CGAGGGT BPK1 NGG 20GGCGAGGGCG 25. GGCGAGGGCGATGCCA 26. 353 site 4 ATGCCACCTA CCTACGGC MSPNGG 20 GGTCGCCACC 27. GGTCGCCACCATGGTGA 28. 792 site 5 ATGGTGAGCAGCAAGGG MSP NGG 20 GGTCAGGGTG 29. GGTCAGGGTGGTCACGA 30. 795 site 6GTCACGAGGG GGGTGGG FYF1 NGG 20 GGTGGTGCAG 31. GGTGGTGCAGATGAACT 32. 328site 7 ATGAACTTCA TCAGGGT JAF1 NGG 17 GGTGCAGATG 33. GGTGCAGATGAACTTCA34. 001 site 7 AACTTCA GGGT BPK1 NGG 20 GTTGGGGTCTT 35.GTTGGGGTCTTTGCTCA 36. 365 site 8 TGCTCAGGG GGGCGGA MSP NGG 20 GGTGGTCACG37. GGTGGTCACGAGGGTGG 38. 794 site 9 AGGGTGGGCC GCCAGGG FYF1 NGG 20GATGCCGTTCT 39. GATGCCGTTCTTCTGCTT 40. 327 site 10 TCTGCTTGT GTCGGC JAF9NGG 17 GCCGTTCTTCT 41. GCCGTTCTTCTGCTTGTC 42. 97 site 10 GCTTGT GGC BPK1NGG 20 GTCGCCACCA 43. GTCGCCACCATGGTGAG 44. 347 site 11 TGGTGAGCAACAAGGGC BPK1 NGG 20 GCACTGCACG 45. GCACTGCACGCCGTAGG 46. 369 site 12CCGTAGGTCA TCAGGGT MSP NGG 20 GTGAACCGCA 47. GTGAACCGCATCGAGCT 48. 2545site 13 TCGAGCTGAA GAAGGGC MSP NGG 20 GAAGGGCATC 49. GAAGGGCATCGACTTCA50. 2546 site 14 GACTTCAAGG AGGAGGA MSP NGG 20 GCTTCATGTGG 51.GCTTCATGTGGTCGGGG 52. 2547 site 15 TCGGGGTAG TAGCGGC MSP NGG 20GCTGAAGCAC 53. GCTGAAGCACTGCACGC 54. 2548 site 16 TGCACGCCGT CGTAGGT MSPNGG 20 GCCGTCGTCCT 55. GCCGTCGTCCTTGAAGA 56. 2549 site 17 TGAAGAAGAAGATGGT MSP NGG 20 GACCAGGATG 57. GACCAGGATGGGCACC 58. 2550 site 18GGCACCACCC ACCCCGGT MSP NGG 20 GACGTAGCCT 59. GACGTAGCCTTCGGGCA 60. 2551site 19 TCGGGCATGG TGGCGGA MSP NGG 20 GAAGTTCGAG 61. GAAGTTCGAGGGCGAC62. 2553 site 20 GGCGACACCC ACCCTGGT MSP NGG 20 GAGCTGGACG 63.GAGCTGGACGGCGACGT 64. 2554 site 21 GCGACGTAAA AAACGGC MSP NGG 20GGCATCGCCC 65. GGCATCGCCCTCGCCCT 66. 2555 site 22 TCGCCCTCGC CGCCGGA MSPNGG 20 GGCCACAAGT 67. GGCCACAAGTTCAGCGT 68. 2556 site 23 TCAGCGTGTCGTCCGGC FYF1 NGG 20 GGGCGAGGAG 69. GGGCGAGGAGCTGTTCA 70. 331 site 24CTGTTCACCG CCGGGGT FYF1 NGG 18 GCGAGGAGCT 71. GCGAGGAGCTGTTCACC 72. 560site 24 GTTCACCG GGGGT BPK1 NGG 20 CCTCGAACTTC 73. CCTCGAACTTCACCTCG 74.348 site 25- ACCTCGGCG GCGCGGG no 5′ G BPK1 NGG 20 GCTCGAACTTC 75.GCTCGAACTTCACCTCG 76. 349 site 25- ACCTCGGCG GCGCGGG mm 5′ G BPK1 NGG 20CAACTACAAG 77. CAACTACAAGACCCGCG 78. 351 site 26- ACCCGCGCCG CCGAGGT no5′ G BPK1 NGG 20 GAACTACAAG 79. GAACTACAAGACCCGCG 80. 352 site 26-ACCCGCGCCG CCGAGGT mm 5′ G BPK1 NGG 20 CGCTCCTGGA 81. CGCTCCTGGACGTAGCC82. 373 site 27- CGTAGCCTTC TTCGGGC no 5′ G BPK1 NGG 20 GGCTCCTGGA 83.CGCTCCTGGACGTAGCC 84. 375 site 27- mm 5′ CGTAGCCTTC TTCGGGC G BPK1 NGG20 AGGGCGAGGA 85. AGGGCGAGGAGCTGTTC 86. 377 site 28- GCTGTTCACC ACCGGGGno 5′ G BPK1 NGG 20 GGGGCGAGGA 87. GGGGCGAGGAGCTGTTC 88. 361 site 28-GCTGTTCACC ACCGGGG mm 5′ G BPK1 NGAA 20 GTTCGAGGGC 89. GTTCGAGGGCGACACCC90. 468 site 1 GACACCCTGG TGGTGAA MSP NGAA 20 GTTCACCAGG 91.GTTCACCAGGGTGTCGC 92. 807 site 2 GTGTCGCCCT CCTCGAA MSP NGAC 20GCCCACCCTC 93. GCCCACCCTCGTGACCA 94. 170 site 1 GTGACCACCC CCCTGAC MSPNGAC 20 GCCCTTGCTCA 95. GCCCTTGCTCACCATGG 96. 790 site 2 CCATGGTGGTGGCGAC MSP NGAT 20 GTCGCCGTCC 97. GTCGCCGTCCAGCTCGA 98. 171 site 1AGCTCGACCA CCAGGAT MSP NGAT 20 GTGTCCGGCG 99. GTGTCCGGCGAGGGCGA 100. 169site 2 AGGGCGAGGG GGGCGAT MSP NGAG 20 GGGGTGGTGC 101. GGGGTGGTGCCCATCCT102. 168 site 1 CCATCCTGGT GGTCGAG MSP NGAG 20 GCCACCATGG 103.GCCACCATGGTGAGCAA 104. 366 site 2 TGAGCAAGGG GGGCGAG Endogenous genesFYF15 NGG 20 GAGTCCGAGC 105. GAGTCCGAGCAGAAG 106. 48 site 1 AGAAGAAGAAAGAAGGGC A MSP80 NGG 20 GTCACCTCCA 107. GTCACCTCCAATGACT 108. 9 site 2ATGACTAGGG AGGGTGGG VC475 NGG 20 GGGAAGACTG 109. GGGAAGACTGAGGCT 110.site 3 AGGCTACATA ACATAGGGT MSP81 NGA 20 GCCACGAAGC 111. GCCACGAAGCAGGCC112. 4 *1 site 1 AGGCCAATGG AATGGGGAG FANCF DR348 NGG 20 GGAATCCCTT 113.GGAATCCCTTCTGCAG 114. site 1 CTGCAGCACC CACCTGGA MSP81 NGG 20 GCTGCAGAAG115. GCTGCAGAAGGGATTC 116. 5 site 2 GGATTCCATG CATGAGGT MSP81 NGG 20GGCGGCTGCA 117. GGCGGCTGCACAACCA 118. 6 site 3 CAACCAGTGG GTGGAGGC MSP81NGG 20 GCTCCAGAGC 119. GCTCCAGAGCCGTGCG 120. 7 site 4 CGTGCGAATGAATGGGGC MSP81 NGA GAATCCCTTC 121. GAATCCCTTCTGCAGC 122. 8 *2 site 1 20TGCAGCACCT ACCTGGAT MSP82 NGA 20 GCGGCGGCTG 123. GCGGCGGCTGCACAAC 124. 0*3 site 2 CACAACCAGT CAGTGGAG MSP88 NGA 20 GGTTGTGCAG 125.GGTTGTGCAGCCGCCG 126. 5 *4 site 3 CCGCCGCTCC CTCCAGAG RUNX1 MSP82 NGG 20GCATTTTCAG 127. GCATTTTCAGGAGGAA 128. 2 site 1 GAGGAAGCGA GCGATGGC MSP82NGG 20 GGGAGAAGA 129. GGGAGAAGAAAGAGA 130. 5 site 2 AAGAGAGATG GATGTAGGGT MSP82 NGA 20 GGTGCATTTT 131. GGTGCATTTTCAGGAG 132. 6 *5 site 1CAGGAGGAAG GAAGCGAT MSP82 NGA 20 GAGATGTAGG 133. GAGATGTAGGGCTAGA 134. 8*6 site 2 GCTAGAGGGG GGGGTGAG MSP17 NGAA 20 GGTATCCAGC 135.GGTATCCAGCAGAGGG 136. 25 site 1 AGAGGGGAG GAGAAGAA MSP17 NGAA 20GAGGCATCTC 137. GAGGCATCTCTGCACC 138. 26 site 2 TGCACCGAGG GAGGTGAAMSP17 NGAC 20 GAGGGGT GAG 139. GAGGGGTGAGGCTGA 140. 28 site 1 GCTGAAACAGAACAGTGAC MSP17 NGAC 20 GAGCAAAAGT 141. GAGCAAAAGTAGATAT 142. 30 site 2AGATATTACA TACAAGAC MSP17 NGAT 20 GGAATTCAAA 143. GGAATTCAAACTGAGG 144.32 site 1 CT GAGGCATA CATATGAT MSP82 NGAT 20 GCAGAGGGGA 145.GCAGAGGGGAGAAGA 146. 9 site 2 GAAGAAAGA AAGAGAGAT G MSP17 NGAG 20GCACCGAGGC 147. GCACCGAGGCATCTCT 148. 34 site 1 ATCTCTGCAC GCACC GAGMSP82 NGAG 20 GAGATGTAGG 149. GAGATGTAGGGCTAGA 150. 8 site 2 GCTAGAGGGGGGGGTGAG ZSCAN2 NN675 NGG 20 GTGCGGCAAG 151. GTGCGGCAAGAGCTTC 152. siteAGCTTCAGCC AGCCGGGG VEGFA VC297 NGG 20 GGGTGGGGGG 153. GGGTGGGGGGAGTTTG154. site 1 AGTTTGCTCC CTCCTGGA VC299 NGG 20 GACCCCCTCC 155.GACCCCCTCCACCCCG 156. site 2 ACCCCGCCTC CCTCCGGG VC228 NGG 20 GGTGAGTGAG157. GGTGAGTGAGTGTGTG 158. site 3 TGTGTGCGTG CGTGTGGG BPK18 NGA 20GCGAGCAGCG 159. GCGAGCAGCGTCTTCG 160. 46 *7 site 1 TCTTCGAGAG AGAGTGAGZNF629 NN675 NGA 20 GTGCGGCAAG 161. GTGCGGCAAGAGCTTC 162. *8 siteAGCTTCAGCC AGCCAGAG *1, NGA EMX1 site 4 from Kleinstiver et al., Nature2015 *2, NGA FANCF site 1 from Kleinstiver et al., Nature 2015 *3, NGAFANCF site 3 from Kleinstiver et al., Nature 2015 *4, NGA FANCF site 4from Kleinstiver et al., Nature 2015 *5, NGA RUNX1 site 1 fromKleinstiver et al., Nature 2015 *6, NGA RUNX1 site 3 from Kleinstiver etal., Nature 2015 *7, NGA VEGFA site 1 from Kleinstiver et al., Nature2015 *8, NGA ZNF629 site from Kleinstiver et al., Nature 2015Genome-Wide Specificity of SpCas9-HF1

To test whether SpCas9-HF1 exhibited reduced off-target effects in humancells, the genome-wide unbiased identification of double-stranded breaksenabled by sequencing (GUIDE-seq) method was used. GUIDE-seq usesintegration of a short double-stranded oligodeoxynucleotide (dsODN) taginto double-strand breaks to enable amplification and sequencing ofadjacent genomic sequence, with the number of tag integrations at anygiven site providing a quantitative measure of cleavage efficiency(Tsai, S. Q. et al,Nat Biotechnol 33, 187-197 (2015)). GUIDE-seq wasused to compare the spectrum of off-target effects induced by wild-typeSpCas9 and SpCas9-HF1 using eight different sgRNAs targeted to varioussites in the endogenous human EMX1, FANCF, RUNX1, and ZSCAN2 genes. Thesequences targeted by these sgRNAs are unique and have variable numbersof predicted mismatched sites in the reference human genome (Table 2).Assessment of on-target dsODN tag integration (by restriction fragmentlength polymorphism (RFLP) assay) and indel formation (by T7EI assay)for the eight sgRNAs revealed comparable on-target activities withwild-type SpCas9 and SpCas9-HF1 (FIGS. 7a and 7b , respectively).GUIDE-seq experiments showed that seven of the eight sgRNAs inducedcleavage at multiple genome-wide off-target sites (ranging from 2 to 25per sgRNA) with wild-type SpCas9, whereas the eighth sgRNA (for FANCFsite 4) did not produce any detectable off-target sites (FIGS. 2a and 2b). However, six of the seven sgRNAs that induced indels with wild-typeSpCas9 showed a strikingly complete absence of GUIDE-seq detectableoff-target events with SpCas9-HF1 (FIGS. 2a and 2b ); and the remainingseventh sgRNA (for FANCF site 2) induced only a single detectablegenome-wide off-target cleavage event, at a site harboring one mismatchwithin the protospacer seed sequence (FIG. 2a ). Collectively, theoff-target sites that were not detected when using SpCas9-HF1 harboredone to six mismatches in the protospacer and/or PAM sequence (FIG. 2c ).As with wild-type SpCas9, the eighth sgRNA (for FANCF site 4) did notyield any detectable off-target cleavage events when tested withSpCas9-HF1 (FIG. 2a ).

To confirm the GUIDE-seq findings, targeted amplicon sequencing was usedto more directly measure the frequencies of NHEJ-mediated indelmutations induced by wild-type SpCas9 and SpCas9-HF1. For theseexperiments, human cells were transfected only with sgRNA- andCas9-encoding plasmids (i.e., without the GUIDE-seq tag).Next-generation sequencing was then used to examine 36 of the 40off-target sites that had been identified with wild-type SpCas9 for sixsgRNAs in the GUIDE-seq experiments (four of the 40 sites could not beexamined because they could not be specifically amplified from genomicDNA). These deep sequencing experiments showed that: (1) wild-typeSpCas9 and SpCas9-HF1 induced comparable frequencies of indels at eachof the six sgRNA on-target sites (FIGS. 3a and 3b ); (2) wild-typeSpCas9, as expected showed statistically significant evidence of indelmutations at 35 of the 36 off-target sites (FIG. 3b ) at frequenciesthat correlated well with GUIDE-seq read counts for these same sites(FIG. 3c ); and (3) the frequencies of indels induced by SpCas9-HF1 at34 of the 36 off-target sites were indistinguishable from the backgroundlevel of indels observed in samples from control transfections (FIG. 3b). For the two off-target sites that appeared to have statisticallysignificant mutation frequencies with SpCas9-HF1 relative to thenegative control, the mean frequencies of indels were 0.049% and 0.037%,levels at which it is difficult to determine whether these are due tosequencing/PCR error or are bona fide nuclease-induced indels. Based onthese results, it was concluded that SpCas9-HF1 can completely or nearlycompletely reduce off-target mutations that occur across a range ofdifferent frequencies with wild-type SpCas9 to undetectable levels.

Next the capability of SpCas9-HF1 to reduce genome-wide off-targeteffects of sgRNAs that target atypical homopolymeric or repetitivesequences was assessed. Although many now try to avoid on-target siteswith these characteristics due to their relative lack of orthogonalityto the genome, it was desirable to explore whether SpCas9-HF1 mightreduce off-target indels even for these challenging targets. Therefore,previously characterized sgRNAs (Fu, Y. et al., Nat Biotechnol 31,Tsai,S. Q. et al., Nat Biotechnol 33, 187-197 (2015) were used that targeteither a cytosine-rich homopolymeric sequence or a sequence containingmultiple TG repeats in the human VEGFA gene (VEGFA site 2 and VEGFA site3, respectively) (Table 2). In control experiments, each of these sgRNAsinduced comparable levels of GUIDE-seq ds ODN tag incorporation (FIG. 7c) and indel mutations (FIG. 7d ) with both wild-type SpCas9 andSpCas9-HF1, demonstrating that SpCas9-HF1 was not impaired in on-targetactivity with either of these sgRNAs. Importantly, GUIDE-seq experimentsrevealed that SpCas9-HF1 was highly effective at reducing off-targetsites of these sgRNAs, with 123/144 sites for VEGFA site 2 and 31/32sites for VEGFA site 3 not detected (FIGS. 4a and 4b ). Examination ofthese off-target sites not detected with SpCas9-HF1 showed that theyeach possessed a range of total mismatches within their protospacer andPAM sequences: 2 to 7 mismatches for the VEGFA site 2 sgRNA and 1 to 4mismatches for the VEGFA site 3 sgRNA (FIG. 4c ); also, nine of theseoff-targets for VEGFA site 2 may have a potential bulged base (Lin, Y.et al,. Nucleic Acids Res 42, 7473-7485 (2014).at the sgRNA-DNAinterface (FIG. 4a and FIG. 8). The sites that were not detected withSpCas9-HF1 possessed 2 to 6 mismatches for the VEGFA site 2 sgRNA and 2mismatches in the single site for the VEGFA site 3 sgRNA (FIG. 4c ),with three off-target sites for VEGFA site 2 sgRNA again having apotential bulge (FIG. 8). Collectively, these results demonstrated thatSpCas9-HF1 can be highly effective at reducing off-target effects ofsgRNAs targeted to simple repeat sequences and can also have substantialimpacts on sgRNAs targeted to homopolymeric sequences.

TABLE 21 Summary of potential mismatched sites in the reference humangenome for the ten sgRNAs examined by GUIDE-seq mismatches to on-targetsite* site spacer with PAM 1 2 3 4 5 6 total EMX1-1GAGTCCGAGCAGAAGAAGAAGGG (SEQ ID NO: 163) 0 1 18 273 2318 15831 18441EMX1-2 GTCACCTCCAATGACTAGGGTGG (SEQ ID NO: 164) 0 0 3 68 780 6102 6953FANCF-1 GGAATCCCTTCTGCAGCACCTGG (SEQ ID NO: 165) 0 1 18 288 1475 961111393 FANCF-2 GCTGCAGAAGGGATTCCATGAGG (SEQ ID NO: 166) 1 1 29 235 200013047 15313 FANCF-3 GGCGGCTGCACAACCAGTGGAGG (SEQ ID NO: 167) 0 0 11 79874 6651 7615 FANCF-4 GCTCCAGAGCCGTGCGAATGGGG (SEQ ID NO: 168) 0 0 6 59639 5078 5782 RUNX1-1 GCATTTTCAGGAGGAAGCGATGG(SEQ ID NO: 169) 0 2 6 1891644 11546 13387 ZSCAN2 GTGCGGCAAGAGCTTCAGCCGGG(SEQ ID NO: 170) 0 3 12127 1146 10687 11975 VEGFA2 GACCCCCTCCACCCCGCCTCCGG(SEQ ID NO: 171) 0 235 456 3905 17576 21974 VEGFA3 GGTGAGTGAGTGTGTGCGTGTGG (SEQ ID NO: 172)1 17 383 6089 13536 35901 55927 *determined using Cas-OFFinder (Bae etal., Bioinformatics 30, 1473-1475 (2014))

TABLE 4 Oligonucleotides used in the study SEQ ID sequence NO:description of T7E1 primers forward primer to amplify EMX1 inGGAGCAGCTGGTCAG 173. U2OS human cells AGGGG reverse primer to amplifyEMX1 in CCATAGGGAAGGGGG 174. U2OS human cells ACACTGG forward primer toamplify FANCF in GGGCCGGGAAAGAGT 175. U2OS human cells TGCTG reverseprimer to amplify FANCF in GCCCTACATCTGCTCT 176. U2OS human cells CCCTCCforward primer to amplify RUNX1 in CCAGCACAACTTACTC 177. U2OS humancells GCACTTGAC reverse primer to amplify RUNX1 in CATCACCAACCCACAG 178.U2OS human cells CCAAGG forward primer to amplify VEGFA inTCCAGATGGCACATTG 179. U2OS human cells TCAG reverse primer to amplifyVEGFA in AGGGAGCAGGAAAGT 180. U2OS human cells GAGGT forward primer toamplify VEGFA CGAGGAAGAGAGAGA 181. (NGG site 2) in U2OS human cellsCGGGGTC reverse primer to amplify VEGFA CTCCAATGCACCCAAG 182. (NGG site2) in U2OS human cells ACAGCAG forward primer to amplify ZSCAN2AGTGTGGGGTGTGTGG 183. in U2OS human cells GAAG reverse primer to amplifyZSCAN2 GCAAGGGGAAGACTC 184. in U2OS human cells TGGCA forward primer toamplify ZNF629 TACGAGTGCCTAGAGT 185. in U2OS human cells GCG reverseprimer to amplify ZNF629 GCAGATGTAGGTCTTG 186. in U2OS human cellsGAGGAC description of deep sequencing primers forward primer to amplifyEMX1-1 GGAGCAGCTGGTCAG 187. on-target AGGGG reverse primer to amplifyEMX1-1 CGATGTCCTCCCCATT 188. on-target GGCCTG forward primer to amplifyEMX1-1- GTGGGGAGATTTGCAT 189. GUIDE_seq-OT#1 CTGTGGAGG reverse primer toamplify EMX1-1- GCTTTTATACCATCTT 190. GUIDE_seq-OT#1 GGGGTTACAG forwardprimer to amplify EMX1-1- CAATGTGCTTCAACCC 191. GUIDE_seq-OT#2 ATCACGGCreverse primer to amplify EMX1-1- CCATGAATTTGTGATG 192. GUIDE_seq-OT#2GATGCAGTCTG forward primer to amplify EMX1-1- GAGAAGGAGGTGCAG 193.GUIDE_seq-OT#3 GAGCTAGAC reverse primer to amplify EMX1-1-CATCCCGACCTTCATC 194. GUIDE_seq-OT#3 CCTCCTGG forward primer to amplifyEMX1-1- GTAGTTCTGACATTCC 195. GUIDE_seq-OT#4 TCCTGAGGG reverse primer toamplify EMX1-1- TCAAACAAGGTGCAG 196. GUIDE_seq-OT#4 ATACAGCA forwardprimer to amplify EMX1-1- CAGGGTCGCTCAGTCT 197. GUIDE_seq-OT#5 GTGTGGreverse primer to amplify EMX1-1- CCAGCGCACCATTCAC 198. GUIDE_seq-OT#5TCCACCTG forward primer to amplify EMX1-1- GGCTGAAGAGGAAGA 199.GUIDE_seq-OT#6 CCAGACTCAG reverse primer to amplify EMX1-1-GGCCCCTCTGAATTCA 200. GUIDE_seq-OT#6 ATTCTCTGC forward primer to amplifyEMX1-1- CCACAGCGAGGAGTG 201. GUIDE_seq-OT#7 ACAGCC reverse primer toamplify EMX1-1- CCAAGTCTTTCCTAAC 202. GUIDE_seq-OT#7 TCGACCTTGG forwardprimer to amplify EMX1-1- CCCTAGGCCCACACCA 203. GUIDE_seq-OT#8 GCAATGreverse primer to amplify EMX1-1- GGGATGGGAATGGGA 204. GUIDE_seq-OT#8ATGTGAGGC forward primer to amplify EMX1-2 GCCCAGGTGAAGGTGT 205.on-target GGTTCC reverse primer to amplify EMX1-2 CCAAAGCCTGGCCAGG 206.on-target GAGTG forward primer to amplify EMX1-2- AGGCAAAGATCTAGG 207.GUIDE_seq-OT#1 ACCTGGATGG reverse primer to amplify EMX1-2-CCATCTGAGTCAGCCA 208. GUIDE_seq-OT#1 GCCTTGTC forward primer to amplifyEMX1-2- GGTTCCCTCCCTTCTG 209. GUIDE_seq-OT#2 AGCCC reverse primer toamplify EMX1-2- GGATAGGAATGAAGA 210. GUIDE_seq-OT#2 CCCCCTCTCC forwardprimer to amplify EMX1-2- GGACTGGCTGGCTGTG 211. GUIDE_seq-OT#3 TGTTTTGAGreverse primer to amplify EMX1-2- CTTATCCAGGGCTACC 212. GUIDE_seq-OT#3TCATTGCC forward primer to amplify EMX1-2- GCTGCTGCTGCTTTGA 213.GUIDE_seq-OT#4 TCACTCCTG reverse primer to amplify EMX1-2-CTCCTTAAACCCTCAG 214. GUIDE_seq-OT#4 AAGCTGGC forward primer to amplifyEMX1-2- GCACTGTCAGCTGATC 215. GUIDE_seq-OT#5 CTACAGG reverse primer toamplify EMX1-2- ACGTTGGAACAGTCGA 216. GUIDE_seq-OT#5 GCTGTAGC forwardprimer to amplify EMX1-2- TGTGCATAACTCATGT 217. GUIDE_seq-OT#6 TGGCAAACTreverse primer to amplify EMX1-2- TCCACAACTACCCTCA 218. GUIDE_seq-OT#6GCTGGAG forward primer to amplify EMX1-2- CCACTGACAATTCACT 219.GUIDE_seq-OT#7 CAACCCTGC reverse primer to amplify EMX1-2-AGGCAGACCAGTTATT 220. GUIDE_seq-OT#7 TGGCAGTC forward primer to amplifyEMX1-2- ACAGGCGCAGTTCACT 221. GUIDE_seq-OT#9 GAGAAG reverse primer toamplify EMX1-2- GGGTAGGCTGACTTTG 222. GUIDE_seq-OT#9 GGCTCC forwardprimer to amplify FANCF-1 GCCCTCTTGCCTCCAC 223. on-target TGGTTG reverseprimer to amplify FANCF-1 CGCGGATGTTCCAATC 224. on-target AGTACGCforward primer to amplify FANCF-1- GCGGGCAGTGGCGTCT 225. GUIDE_seq-OT#1TAGTCG reverse primer to amplify FANCF-1- CCCTGGGTTTGGTTGG 226.GUIDE_seq-OT#1 CTGCTC forward primer to amplify FANCF-1-CTCCTTGCCGCCCAGC 227. GUIDE_seq-OT#2 CGGTC reverse primer to amplifyFANCF-1- CACTGGGGAAGAGGC 228. GUIDE_seq-OT#2 GAGGACAC forward primer toamplify FANCF-1- CCAGTGTTTCCCATCC 229. GUIDE_seq-OT#3 CCAACAC reverseprimer to amplify FANCF-1- GAATGGATCCCCCCCT 230. GUIDE_seq-OT#3 AGAGCTCforward primer to amplify FANCF-1- CAGGCCCACAGGTCCT 231. GUIDE_seq-OT#4TCTGGA reverse primer to amplify FANCF-1- CCACACGGAAGGCTG 232.GUIDE_seq-OT#4 ACCACG forward primer to amplify FANCF-3 GCGCAGAGAGAGCAG233. on-target GACGTC reverse primer to amplify FANCF-3 GCACCTCATGGAATCC234. on-target CTTCTGC forward primer to amplify FANCF-3-CAAGTGATGCGACTTC 235. GUIDE_seq-OT#1 CAACCTC reverse primer to amplifyFANCF-3- CCCTCAGAGTTCAGCT 236. GUIDE_seq-OT#1 TAAAAAGACC forward primerto amplify FANCF-3- TGCTTCTCATCCACTCT 237. GUIDE_seq-OT#2 AGACTGCTreverse primer to amplify FANCF-3- CACCAACCAGCCATGT 238. GUIDE_seq-OT#2GCCATG forward primer to amplify FANCF-3- CTGCCTGTGCTCCTCG 239.GUIDE_seq-OT#3 ATGGTG reverse primer to amplify FANCF-3-GGGTTCAAAGCTCATC 240. GUIDE_seq-OT#3 TGCCCC forward primer to amplifyFANCF-3- GCATGTGCCTTGAGAT 241. GUIDE_seq-OT#4 TGCCTGG reverse primer toamplify FANCF-3- GACATTCAGAGAAGC 242. GUIDE_seq-OT#4 GACCATGTGG forwardprimer to amplify FANCF-3- CCATCTTCCCCTTTGG 243. GUIDE_seq-OT#5 CCCACAGreverse primer to amplify FANCF-3- CCCCAAAAGTGGCCAA 244. GUIDE_seq-OT#5GAGCCTGAG forward primer to amplify FANCF-3- GTTCTCCAAAGGAAGA 245.GUIDE_seq-OT#6 GAGGGGAATG reverse primer to amplify FANCF-3-GGTGCTGTGTCCTCAT 246. GUIDE_seq-OT#6 GCATCC forward primer to amplifyFANCF-3- CGGCTTGCCTAGGGTC 247. GUIDE_seq-OT#7 GTTGAG reverse primer toamplify FANCF-3- CCTTCAGGGGCTCTTC 248. GUIDE_seq-OT#7 CAGGTC forwardprimer to amplify RUNX1-1 GGGAACTGGCAGGCA 249. on-target CCGAGG reverseprimer to amplify RUNX1-1 GGGTGAGGCTGAAAC 250. on-target AGTGACC forwardprimer to amplify RUNX1-1- GGGAGGATGTTGGTTT 251. GUIDE_seq-OT#1TAGGGAACTG reverse primer to amplify RUNX1-1- TCCAATCACTACATGC 252.GUIDE_seq-OT#1 CATTTTGAAGA forward primer to amplify RUNX1-1-CCACCCTCTTCCTTTG 253. GUIDE_seq-OT#2 ATCCTCCC reverse primer to amplifyRUNX1-1- TCCTCCCTACTCCTTCA 254. GUIDE_seq-OT#2 CCCAGG forward primer toamplify ZSCAN2 GAGTGCCTGACATGTG 255. on-target GGGAGAG reverse primer toamplify ZSCAN2 TCCAGCTAAAGCCTTT 256. on-target CCCACAC forward primer toamplify ZSCAN2- GAACTCTCTGATGCAC 257. GUIDE_seq-OT#1 CTGAAGGCTG reverseprimer to amplify ZSCAN2- ACCGTATCAGTGTGAT 258. GUIDE_seq-OT#1 GCATGTGGTforward primer to amplify ZSCAN2- TGGGTTTAATCATGTG 259. GUIDE_seq-OT#2TTCTGCACTATG reverse primer to amplify ZSCAN2- CCCATCTTCCATTCTG 260.GUIDE_seq-OT#2 CCCTCCAC forward primer to amplify ZSCAN2-CAGCTAGTCCATTTGT 261. GUIDE_seq-OT#3 TCTCAGACTGTG reverse primer toamplify ZSCAN2- GGCCAACATTGTGAAA 262. GUIDE_seq-OT#3 CCCTGTCTC forwardprimer to amplify ZSCAN2- CCAGGGACCTGTGCTT 263. GUIDE_seq-OT#4 GGGTTCreverse primer to amplify ZSCAN2- CACCCCATGACCTGGC 264. GUIDE_seq-OT#4ACAAGTG forward primer to amplify ZSCAN2- AAGTGTTCCTCAGAAT 265.GUIDE_seq-OT#5 GCCAGCCC reverse primer to amplify ZSCAN2-CAGGAGTGCAGTTGTG 266. GUIDE_seq-OT#5 TTGGGAG forward primer to amplifyZSCAN2- CTGATGAAGCACCAGA 267. GUIDE_seq-OT#6 GAACCCACC reverse primer toamplify ZSCAN2- CACACCTGGCACCCAT 268. GUIDE_seq-OT#6 ATGGC forwardprimer to amplify ZSCAN2- GATCCACACTGGTGAG 269. GUIDE_seq-OT#7 AAGCCTTACreverse primer to amplify ZSCAN2- CTTCCCACACTCACAG 270. GUIDE_seq-OT#7CAGATGTAGG

Refining the Specificity of SpCas9-HF1

Previously described methods such as truncated gRNA5 (Fu, Y. et al., NatBiotechnol 32, 279-284 (2014)) and the SpCas9-D1135E variant(Kleinstiver, B. P. et al., Nature 523, 481-485 (2015)) can partiallyreduce SpCas9 off-target effects, and the present inventors wonderedwhether these might be combined with SpCas9-HF1 to further improve itsgenome-wide specificity. Testing of SpCas9-HF1 with matched full-lengthand truncated sgRNAs targeted to four sites in the human cell-based EGFPdisruption assay revealed that shortening sgRNA complementarity lengthsubstantially impaired on-target activities (FIG. 9). By contrast,SpCas9-HF1 with an additional D1135E mutation (a variant referred toherein as SpCas9-HF2) retained 70% or more activity of wild-type SpCas9with six of eight sgRNAs tested using a human cell-based EGFP disruptionassay (FIGS. 5a and 5b ). SpCas9-HF3 and SpCas9-HF4 variants were alsocreated harboring L169A or Y450A mutations, respectively, at positionswhose side chains mediated hydrophobic non-specific interactions withthe target DNA on its PAM proximal end (Nishimasu, H. et al., Cell 156,935-949 (2014); Jiang, F., et al., Science 348, 1477-1481 (2015)).SpCas9-HF3 and SpCas9-HF4 retained 70% or more of the activitiesobserved with wild-type SpCas9 with the same six out of eightEGFP-targeted sgRNAs (FIGS. 5a and 5b ).

To determine whether SpCas9-HF2, -HF3, and -HF4 could reduce indelfrequencies at two off-target sites (for the FANCF site 2 and VEGFA site3 sgRNAs) that were resistant to SpCas9-HF1, further experiments wereperformed. For the FANCF site 2 off-target, which bears a singlemismatch in the seed sequence of the protospacer, SpCas9-HF4 reducedindel mutation frequencies to near background level as judged by T7EIassay while also beneficially increasing on-target activity (FIG. 5c ),resulting in the greatest increase in specificity among the threevariants (FIG. 5d ). For the VEGFA site 3 off-target site, which bearstwo protospacer mismatches (one in the seed sequence and one at thenucleotide most distal from the PAM sequence), SpCas9-HF2 showed thegreatest reduction in indel formation while showing only modest effectson on-target mutation efficiency (FIG. 5c ), leading to the greatestincrease in specificity among the three variants tested (FIG. 5d ).Taken together, these results demonstrate the potential for reducingoff-target effects that are resistant to SpCas9-HF1 by introducingadditional mutations at other residues that mediate non-specific DNAcontacts or that may alter PAM recognition.

To generalize the T7E1 assay findings described above that showSpCas9-HF4 and SpCas9-HF2 have improved discrimination relative toSpCas9-HF1 against off-targets of the FANCF site 2 and VEGFA site 3sgRNAs, respectively, the genome-wide specificities of these variantswere examined using GUIDE-seq. Using an RFLP assay, it was determinedthat SpCas9-HF4 and SpCas9-HF2 had similar on-target activities toSpCas9-HF1, as assayed by GUIDE-seq tag integration rates (FIG. 13A).When analyzing the GUIDE-seq data, no new off-target sites wereidentified for SpCas9-HF2 or SpCas9-HF4 (FIG. 13B). Compared toSpCas9-HF1, off-target activities at all sites were either renderedundetectable by GUIDE-seq or substantially decreased. Relative toSpCas9-HF1, SpCas9-HF4 had nearly 26-fold better specificity against thesingle FANCF site 2 off-target site that remained recalcitrant to thespecificity improvements of SpCas9-HF1 (FIG. 13b ). SpCas9-HF2 hadnearly 4-fold improved specificity relative to SpCas9-HF1 for thehigh-frequency VEGFA site 3 off-target, while also dramatically reducing(>38-fold) or eliminating GUIDE-seq detectable events at otherlow-frequency off-target sites. Of note, the genomic position of 3 ofthese low frequency sites identified for SpCas9-HF1 are adjacent topreviously characterized background U2OS cell breakpoint hotspots.Collectively, these results suggest that the SpCas9-HF2 and SpCas9-HF4variants can improve the genome-wide specificity of SpCas9-HF1.

SpCas9-HF1 robustly and consistently reduced off-target mutations whenusing sgRNAs designed against standard, non-repetitive target sequences.The two off-target sites that were most resistant to SpCas9-HF1 haveonly one and two mismatches in the protospacer. Together, theseobservations suggest that off-target mutations might be minimized toundetectable levels by using SpCas9-HF1 and targeting non-repetitivesequences that do not have closely related sites bearing one or twomismatches elsewhere in the genome (something that can be easilyaccomplished using existing publicly available software programs (Bae,S., et al, Bioinformatics 30, 1473-1475 (2014)). One parameter thatusers should keep in mind is that SpCas9-HF1 may not be compatible withthe common practice of using a G at the 5′ end of the gRNA that ismismatched to the protospacer sequence. Testing of four sgRNAs bearing a5′ G mismatched to its target site showed three of the four haddiminished activities with SpCas9-HF1 compared to wild-type SpCas9 (FIG.10), perhaps reflecting the ability of SpCas9-HF1 to better discriminatea partially matched site.

Further biochemical work can confirm or clarify the precise mechanism bywhich SpCas9-HF1 achieves its high genome-wide specificity. It does notappear that the four mutations introduced alter the stability orsteady-state expression level of SpCas9 in the cell, because titrationexperiments with decreasing concentrations of expression plasmidssuggested that wild-type SpCas9 and SpCas9-HF1 behaved comparably astheir concentrations are lowered (FIG. 11). Instead, the simplestmechanistic explanation is that these mutations decreased the energeticsof interaction between the Cas9-sgRNA and the target DNA, with theenergy of the complex at a level just sufficient to retain on-targetactivity but lowered it enough to make off-target site cleavageinefficient or non-existent. This mechanism is consistent with thenon-specific interactions observed between the residues mutated and thetarget DNA phosphate backbone in structural data (Nishimasu, H. et al.,Cell 156, 935-949 (2014); Anders, C et. Al., Nature 513, 569-573(2014)). A somewhat similar mechanism has been proposed to explain theincreased specificities of transcription activator-like effectornucleases bearing substitutions at positively charged residues(Guilinger, J. P. et al., Nat Methods 11, 429-435 (2014)).

It was possible that SpCas9-HF1 might also be combined with othermutations that have been shown to alter Cas9 function. For example, anSpCas9 mutant bearing three amino acid substitutions(D1135V/R1335Q/T1337R, also known as the SpCas9-VQR variant), recognizessites with NGAN PAMs (with relative efficiencies forNGAG>NGAT=NGAA>NGAC) (Kleinstiver, B. P. et al, Nature 523, 481-485(2015)) and a recently identified quadruple SpCas9 mutant(D1135V/G1218R/R1335Q/T1337R, referred to as the SpCas9-VRQR variant)has improved activities relative to the VQR variant on sites with NGAH(H=A, C, or T) PAMs (FIG. 12a ). Introduction of the four mutations(N497A/R661A/Q695A/Q926A) from SpCas9-HF1 into SpCas9-VQR andSpCas9-VRQR created SpCas9-VQR-HF1 and SpCas9-VRQR-HF1, respectively.Both HF versions of these nucleases showed on-target activitiescomparable (i.e., 70% or more) to their non-HF counterparts with five ofeight sgRNAs targeted to the EGFP reporter gene and with seven of eightsgRNAs targeted to endogenous human gene sites (FIGS. 12b-12d ).

More broadly, these results illuminate a general strategy for theengineering of additional high-fidelity variants of CRISPR-associatednucleases. Adding additional mutations at non-specific DNA contactingresidues further reduced some of the very small number of residualoff-target sites that persist with SpCas9-HF1. Thus, variants such asSpCas9-HF2, SpCas9-HF3, SpCas9-HF4, and others can be utilized in acustomized fashion depending on the nature of the off-target sequences.Furthermore, success with engineering high-fidelity variants of SpCas9suggests that the approach of mutating non-specific DNA contacts can beextended to other naturally occurring and engineered Cas9 orthologues(Ran, F. A. et al., Nature 520, 186-191 (2015), Esvelt, K. M. et al.,Nat Methods 10, 1116-1121 (2013); Hou, Z. et al., Proc Natl Acad Sci USA(2013); Fonfara, I. et al., Nucleic Acids Res 42, 2577-2590 (2014);Kleinstiver, B. P. et al, Nat Biotechnol (2015).as well as newerCRISPR-associated nucleases (Zetsche, B. et al., Cell 163, 759-771(2015); Shmakov, S. et al., Molecular Cell 60, 385-397). that are beingdiscovered and characterized with increasing frequency.

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SEQ ID NO: 271 - JDS246: CMV-T7-humanSpCas9-NLS-3xFLAGHuman codon optimized S. pyogenes Cas9 in normal font, NLSdouble underlined,3xFLAG tag in bold:ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGTGA

SEQ ID NO: 272 - VP12: CMV-T7-humanSpCas9-HF1(N497A, R661A, Q695A,Q926A)-NLS-3xFLAGHuman codon optimized S. pyogenes Cas9 in normal font, modifiedcodons in lower case, NLS double underlined, 3xFLAG tag in bold:ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCCTGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCgccTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGPAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGAgccTTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGgccCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCgccATCACAAAGCATGTTGCGCAGATACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGTGA

SEQ ID NO: 273 - MSP2135: CMV-T7-humanSpCas9-HF2(N497A, R661A, Q695A,Q926A, D1135E)-NLS-3xFLAGHuman codon optimized S. pyogenes Cas9 in normal font, modifiedcodons in lower case, NLS double underlined, 3xFLAG tag in bold:ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCCTGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCgccTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGPAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGAgccTTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGgccCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCgccATCACAAAGCATGTTGCGCAGATACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCgagAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGTGA

SEQ ID NO: 274 - MSP2133: CMV-T7-humanSpCas9-HF4(Y450A, N497A, R661A,Q695A, Q926A)-NLS-3xFLAGHuman codon optimized S. pyogenes Cas9 in normal font, modifiedcodons in lower case, NLS double underlined, 3xFLAG tag in bold:ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTgccTATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCCTGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCgccTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGAgccTTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGgccCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCgccATCACAAAGCATGTTGCGCAGATACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGTGA

SEQ ID NO: 275 - MSP469: CMV-T7-humanSpCas9-VQR(D1135V, R1335Q,T1337R)-NLS-3xFLAGHuman codon optimized S. pyogenes Cas9 in normal font, modifiedcodons in lower case, NLS double underlined, 3xFLAG tag in bold:ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCgtgAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAAcagTACagaTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGTGA

SEQ ID NO: 276 - MSP2440: CMV-T7-humanSpCas9-VQR-HF1(N497A, R661A,Q695A, Q926A, D1135V, R1335Q, T1337R)-NLS-3xFLAGHuman codon optimized S. pyogenes Cas9 in normal font, modifiedcodons in lower case, NLS double underlined, 3xFLAG tag in bold:ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCCTGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCgccTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGAgccTTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGgccCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCgccATCACAAAGCATGTTGCGCAGATACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCgtgAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAAcagTACagaTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGTGA

SEQ ID NO: 277 - BPK2797: CMV-T7-humanSpCas9-VRQR(D1135V, G1218R,R1335Q, T1337R)-NLS-3xFLAGHuman codon optimized S. pyogenes Cas9 in normal font, modifiedcodons in lower case, NLS double underlined, 3xFLAG tag in bold:ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCgtgAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCagaGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAAcagTACagaTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGTGA

SEQ ID NO: 278 - MSP2443: CMV-T7-humanSpCas9-VRQR-HF1(N497A, R661A,Q695A, Q926A, D1135V, G1218R, R1335Q, T1337R)-NLS-3xFLAGHuman codon optimized S. pyogenes Cas9 in normal font, modifiedcodons in lower case, NLS double underlined, 3xFLAG tag in bold:ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCCTGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCgccTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGAgccTTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGgccCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCgccATCACAAAGCATGTTGCGCAGATACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCgtgAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCagaGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAAcagTACagaTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGTGA

SEQ ID NO: 279 - BPK1520: U6-BsmBIcassette-Sp-sgRNAU6 promoter in normal font, BsmBI sites  italicised , S. pyogenes sgRNAin lower case, U6 terminator double underlined:TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG GAGACG ATTAATG CGTCTCCgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttt

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. An isolated Streptococcus pyogenes Cas9 (SpCas9)protein with mutations at one or both of Q695 and Q926 in SEQ ID NO: 1,and optionally wherein the SpCas9 protein is fused to one or more of anuclear localization sequence, cell penetrating peptide sequence, and/oraffinity tag.
 2. The isolated protein of claim 1, comprising all four ofthe following mutations: N497A, R661A, Q695A, and Q926A.
 3. The isolatedprotein of claim 2 further comprising mutations at one, two, three,four, or all five of L169, Y450, N497, R661, and D1135.
 4. The isolatedprotein of claim 1, comprising mutations at one or both of Q695 andQ926, and optionally one, two, three, four, or all five of L169, Y450,N497, R661, and D1135.
 5. The isolated protein of claim 1, furthercomprising one or more of the following mutations: D1135E; D1135V;D1135V/R1335Q/T1337R (VQR variant): D1135E/R1335Q/T1337R (EQR variant);D1135V/G1218R/R1335Q/T1337R (VRQR variant); orD1135V/G1218R/R1335E/T1337R (VRER variant).
 6. The isolated protein ofclaim 1, further comprising one or more mutations that decrease nucleaseactivity selected from the group consisting of mutations at D10, E762,D839, H983, or D986; and at H840 or N863.
 7. The isolated protein ofclaim 6, wherein the mutations that decrease nuclease activity are: (i)D10A or D10N, and (ii) H840A, H840N, or H840Y.
 8. A fusion proteincomprising the isolated protein of claim 1, fused to a heterologousfunctional domain, with an optional intervening linker, wherein thelinker does not interfere with activity of the fusion protein.
 9. Thefusion protein of claim 8, wherein the heterologous functional domain isa transcriptional activation domain.
 10. The fusion protein of claim 9,wherein the transcriptional activation domain is from VP64 or NF-_(κ)Bp65.
 11. The fusion protein of claim 8, wherein the heterologousfunctional domain is a transcriptional silencer or transcriptionalrepression domain.
 12. The fusion protein of claim 11, wherein thetranscriptional repression domain is a Krueppel-associated box (KRAB)domain, ERF repressor domain (ERD), or mSin3A interaction domain (SID).13. The fusion protein of claim 11, wherein the transcriptional silenceris Heterochromatin Protein 1 (HP1).
 14. The fusion protein of claim 8,wherein the heterologous functional domain is an enzyme that modifiesthe methylation state of DNA.
 15. The fusion protein of claim 14,wherein the enzyme that modifies the methylation state of DNA is a DNAmethyltransferase (DNMT) or a TET protein.
 16. The fusion protein ofclaim 15, wherein the TET protein is TET1.
 17. The fusion protein ofclaim 8, wherein the heterologous functional domain is an enzyme thatmodifies a histone subunit.
 18. The fusion protein of claim 17, whereinthe enzyme that modifies a histone subunit is a histoneacetyltransferase (HAT), histone deacetylase (HDAC), histonemethyltransferase (HMT), or histone demethylase.
 19. The fusion proteinof claim 8, wherein the heterologous functional domain is a biologicaltether.
 20. The fusion protein of claim 19, wherein the biologicaltether is MS2, Csy4 or lambda N protein.
 21. The fusion protein of claim8, wherein the heterologous functional domain is FokI.
 22. An isolatednucleic acid encoding the protein of claim
 1. 23. A vector comprisingthe isolated nucleic acid of claim
 22. 24. A host cell comprising thenucleic acid of claim
 23. 25. A method of altering the genome of a cell,the method comprising expressing in the cell or contacting the cell withthe isolated protein of claim 1, linked to a guide RNA having a regioncomplementary to a selected portion of the genome of the cell, wherebythe genome of the cell is altered.
 26. The method of claim 25, whereinthe isolated protein comprises one or more of a nuclear localizationsequence, cell penetrating peptide sequence, and/or affinity tag.
 27. Amethod of altering a double stranded DNA D (dsDNA) molecule, the methodcomprising contacting the dsDNA molecule with the isolated protein ofclaim 1, linked to a guide RNA having a region complementary to aselected portion of the dsDNA molecule, whereby the dsDNA molecule isaltered.